SummaryNeandertals and Denisovans, an Asian group distantly related to Neandertals, are the closest evolutionary relatives of present-day humans. They are thus of direct relevance for understanding the origin of modern humans and how modern humans differ from their closest relatives. We will generate genome-wide data from a large number of Neandertal and Denisovan individuals from across their geographical and temporal range as well as from other extinct hominin groups which we may discover. This will be possible by automating highly sensitive approaches to ancient DNA extraction and DNA libraries construction that we have developed so that they can be applied to many specimens from many sites in order to identify those that contain retrievable DNA. Whenever possible we will sequence whole genomes and in other cases use DNA capture methods to generate high-quality data from representative parts of the genome. This will allow us to study the population history of Neandertals and Denisovans, elucidate how many times and where these extinct hominins contributed genes to present-day people, and the extent to which modern humans and archaic groups contributed genetically to Neandertals and Denisovans. By retrieving DNA from specimens that go back to the Middle Pleistocene we will furthermore shed light on the early history and origins of Neandertals and Denisovans.

Neandertals and Denisovans, an Asian group distantly related to Neandertals, are the closest evolutionary relatives of present-day humans. They are thus of direct relevance for understanding the origin of modern humans and how modern humans differ from their closest relatives. We will generate genome-wide data from a large number of Neandertal and Denisovan individuals from across their geographical and temporal range as well as from other extinct hominin groups which we may discover. This will be possible by automating highly sensitive approaches to ancient DNA extraction and DNA libraries construction that we have developed so that they can be applied to many specimens from many sites in order to identify those that contain retrievable DNA. Whenever possible we will sequence whole genomes and in other cases use DNA capture methods to generate high-quality data from representative parts of the genome. This will allow us to study the population history of Neandertals and Denisovans, elucidate how many times and where these extinct hominins contributed genes to present-day people, and the extent to which modern humans and archaic groups contributed genetically to Neandertals and Denisovans. By retrieving DNA from specimens that go back to the Middle Pleistocene we will furthermore shed light on the early history and origins of Neandertals and Denisovans.

SummaryFaithful repair of double stranded DNA breaks (DSBs) is essential, as they are at the origin of genome instability, chromosomal translocations and cancer. Cells repair DSBs through different pathways, which can be faithful or mutagenic, and the balance between them at a given locus must be tightly regulated to preserve genome integrity. Although, much is known about DSB repair factors, how the choice between pathways is controlled within the nuclear environment is not understood. We have shown that nuclear architecture and non-random genome organization determine the frequency of chromosomal translocations and that pathway choice is dictated by the spatial organization of DNA in the nucleus. Nevertheless, what determines which pathway is activated in response to DSBs at specific genomic locations is not understood. Furthermore, the impact of 3D-genome folding on the kinetics and efficiency of DSB repair is completely unknown.
Here we aim to understand how nuclear compartmentalization, chromatin structure and genome organization impact on the efficiency of detection, signaling and repair of DSBs. We will unravel what determines the DNA repair specificity within distinct nuclear compartments using protein tethering, promiscuous biotinylation and quantitative proteomics. We will determine how DNA repair is orchestrated at different heterochromatin structures using a CRISPR/Cas9-based system that allows, for the first time robust induction of DSBs at specific heterochromatin compartments. Finally, we will investigate the role of 3D-genome folding in the kinetics of DNA repair and pathway choice using single nucleotide resolution DSB-mapping coupled to 3D-topological maps.
This proposal has significant implications for understanding the mechanisms controlling DNA repair within the nuclear environment and will reveal the regions of the genome that are susceptible to genomic instability and help us understand why certain mutations and translocations are recurrent in cancer

Faithful repair of double stranded DNA breaks (DSBs) is essential, as they are at the origin of genome instability, chromosomal translocations and cancer. Cells repair DSBs through different pathways, which can be faithful or mutagenic, and the balance between them at a given locus must be tightly regulated to preserve genome integrity. Although, much is known about DSB repair factors, how the choice between pathways is controlled within the nuclear environment is not understood. We have shown that nuclear architecture and non-random genome organization determine the frequency of chromosomal translocations and that pathway choice is dictated by the spatial organization of DNA in the nucleus. Nevertheless, what determines which pathway is activated in response to DSBs at specific genomic locations is not understood. Furthermore, the impact of 3D-genome folding on the kinetics and efficiency of DSB repair is completely unknown.
Here we aim to understand how nuclear compartmentalization, chromatin structure and genome organization impact on the efficiency of detection, signaling and repair of DSBs. We will unravel what determines the DNA repair specificity within distinct nuclear compartments using protein tethering, promiscuous biotinylation and quantitative proteomics. We will determine how DNA repair is orchestrated at different heterochromatin structures using a CRISPR/Cas9-based system that allows, for the first time robust induction of DSBs at specific heterochromatin compartments. Finally, we will investigate the role of 3D-genome folding in the kinetics of DNA repair and pathway choice using single nucleotide resolution DSB-mapping coupled to 3D-topological maps.
This proposal has significant implications for understanding the mechanisms controlling DNA repair within the nuclear environment and will reveal the regions of the genome that are susceptible to genomic instability and help us understand why certain mutations and translocations are recurrent in cancer

Max ERC Funding

1 999 750 €

Duration

Start date: 2017-03-01, End date: 2022-02-28

Project acronym3DEpi

ProjectTransgenerational epigenetic inheritance of chromatin states : the role of Polycomb and 3D chromosome architecture

SummaryEpigenetic inheritance entails transmission of phenotypic traits not encoded in the DNA sequence and, in the most extreme case, Transgenerational Epigenetic Inheritance (TEI) involves transmission of memory through multiple generations. Very little is known on the mechanisms governing TEI and this is the subject of the present proposal. By transiently enhancing long-range chromatin interactions, we recently established isogenic Drosophila epilines that carry stable alternative epialleles, defined by differential levels of the Polycomb-dependent H3K27me3 mark. Furthermore, we extended our paradigm to natural phenotypes. These are ideal systems to study the role of Polycomb group (PcG) proteins and other components in regulating nuclear organization and epigenetic inheritance of chromatin states. The present project conjugates genetics, epigenomics, imaging and molecular biology to reach three critical aims.
Aim 1: Analysis of the molecular mechanisms regulating Polycomb-mediated TEI. We will identify the DNA, protein and RNA components that trigger and maintain transgenerational chromatin inheritance as well as their mechanisms of action.
Aim 2: Role of 3D genome organization in the regulation of TEI. We will analyze the developmental dynamics of TEI-inducing long-range chromatin interactions, identify chromatin components mediating 3D chromatin contacts and characterize their function in the TEI process.
Aim 3: Identification of a broader role of TEI during development. TEI might reflect a normal role of PcG components in the transmission of parental chromatin onto the next embryonic generation. We will explore this possibility by establishing other TEI paradigms and by relating TEI to the normal PcG function in these systems and in normal development.
This research program will unravel the biological significance and the molecular underpinnings of TEI and lead the way towards establishing this area of research into a consolidated scientific discipline.

Epigenetic inheritance entails transmission of phenotypic traits not encoded in the DNA sequence and, in the most extreme case, Transgenerational Epigenetic Inheritance (TEI) involves transmission of memory through multiple generations. Very little is known on the mechanisms governing TEI and this is the subject of the present proposal. By transiently enhancing long-range chromatin interactions, we recently established isogenic Drosophila epilines that carry stable alternative epialleles, defined by differential levels of the Polycomb-dependent H3K27me3 mark. Furthermore, we extended our paradigm to natural phenotypes. These are ideal systems to study the role of Polycomb group (PcG) proteins and other components in regulating nuclear organization and epigenetic inheritance of chromatin states. The present project conjugates genetics, epigenomics, imaging and molecular biology to reach three critical aims.
Aim 1: Analysis of the molecular mechanisms regulating Polycomb-mediated TEI. We will identify the DNA, protein and RNA components that trigger and maintain transgenerational chromatin inheritance as well as their mechanisms of action.
Aim 2: Role of 3D genome organization in the regulation of TEI. We will analyze the developmental dynamics of TEI-inducing long-range chromatin interactions, identify chromatin components mediating 3D chromatin contacts and characterize their function in the TEI process.
Aim 3: Identification of a broader role of TEI during development. TEI might reflect a normal role of PcG components in the transmission of parental chromatin onto the next embryonic generation. We will explore this possibility by establishing other TEI paradigms and by relating TEI to the normal PcG function in these systems and in normal development.
This research program will unravel the biological significance and the molecular underpinnings of TEI and lead the way towards establishing this area of research into a consolidated scientific discipline.

SummaryThis project investigates the two-way relationship between spatio-temporal genome organization and coordinated gene regulation, through an approach at the interface between physics, computer science and biology.
In the nucleus, preferred positions are observed from chromosomes to single genes, in relation to normal and pathological cellular states. Evidence indicates a complex spatio-temporal coupling between co-regulated genes: e.g. certain genes cluster spatially when responding to similar factors and transcriptional noise patterns suggest domain-wide mechanisms. Yet, no individual experiment allows probing transcriptional coordination in 4 dimensions (FISH, live locus tracking, Hi-C...). Interpreting such data also critically requires theory (stochastic processes, statistical physics…). A lack of appropriate experimental/analytical approaches is impairing our understanding of the 4D genome.
Our proposal combines cutting-edge single-molecule imaging, signal-theory data analysis and physical modeling to study how genes coordinate in space and time in a single nucleus. Our objectives are to understand (a) competition/recycling of shared resources between genes within subnuclear compartments, (b) how enhancers communicate with genes domain-wide, and (c) the role of local conformational dynamics and supercoiling in gene co-regulation. Our organizing hypothesis is that, by acting on their microenvironment, genes shape their co-expression with other genes.
Building upon my expertise, we will use dual-color MS2/PP7 RNA labeling to visualize for the first time transcription and motion of pairs of hormone-responsive genes in real time. With our innovative signal analysis tools, we will extract spatio-temporal signatures of underlying processes, which we will investigate with stochastic modeling and validate through experimental perturbations. We expect to uncover how the functional organization of the linear genome relates to its physical properties and dynamics in 4D.

This project investigates the two-way relationship between spatio-temporal genome organization and coordinated gene regulation, through an approach at the interface between physics, computer science and biology.
In the nucleus, preferred positions are observed from chromosomes to single genes, in relation to normal and pathological cellular states. Evidence indicates a complex spatio-temporal coupling between co-regulated genes: e.g. certain genes cluster spatially when responding to similar factors and transcriptional noise patterns suggest domain-wide mechanisms. Yet, no individual experiment allows probing transcriptional coordination in 4 dimensions (FISH, live locus tracking, Hi-C...). Interpreting such data also critically requires theory (stochastic processes, statistical physics…). A lack of appropriate experimental/analytical approaches is impairing our understanding of the 4D genome.
Our proposal combines cutting-edge single-molecule imaging, signal-theory data analysis and physical modeling to study how genes coordinate in space and time in a single nucleus. Our objectives are to understand (a) competition/recycling of shared resources between genes within subnuclear compartments, (b) how enhancers communicate with genes domain-wide, and (c) the role of local conformational dynamics and supercoiling in gene co-regulation. Our organizing hypothesis is that, by acting on their microenvironment, genes shape their co-expression with other genes.
Building upon my expertise, we will use dual-color MS2/PP7 RNA labeling to visualize for the first time transcription and motion of pairs of hormone-responsive genes in real time. With our innovative signal analysis tools, we will extract spatio-temporal signatures of underlying processes, which we will investigate with stochastic modeling and validate through experimental perturbations. We expect to uncover how the functional organization of the linear genome relates to its physical properties and dynamics in 4D.

Max ERC Funding

1 499 750 €

Duration

Start date: 2018-04-01, End date: 2023-03-31

Project acronymBiomeRiskFactors

ProjectDiscovering microbiome-based disease risk factors

Researcher (PI)Eran Segal

Host Institution (HI)WEIZMANN INSTITUTE OF SCIENCE

Call DetailsAdvanced Grant (AdG), LS2, ERC-2017-ADG

SummaryIdentifying risk factors for diseases that can be prevented or delayed by early intervention is of major importance, and numerous genetic, lifestyle, anthropometric and clinical risk factors were found for many different diseases. Another source of potentially pertinent disease risk factors is the human microbiome - the collective genome of trillions of bacteria, viruses, fungi, and parasites that reside in the human gut. However, very few microbiome disease markers were found to date.
Here, we aim to develop risk prediction tools based on the human microbiome that predict the likelihood of an individual to develop a particular condition or disease within 5-10 years. We will use a cohort of >2200 individuals that my group previously assembled, for whom we have clinical profiles, gut microbiome data, and banked blood and stool samples. We will invite people 5-10 years after their initial recruitment time, profile disease status and blood markers, and develop algorithms for predicting 5-10 year onset of Type 2 diabetes, cardiovascular disease, and obesity, using microbiome data from recruitment time.
To increase the likelihood of finding microbiome markers predictive of disease onset, we will develop novel experimental and computational methods for in-depth characterization of microbial gene function, the metabolites produced by the microbiome, the underexplored fungal microbiome members, and the interactions between the gut microbiota and the host adaptive immune system. We will then apply these methods to >2200 banked samples from cohort recruitment time and use the resulting data in devising our microbiome-based risk prediction tools. In themselves, these novel assays and their application to >2200 samples should greatly advance the microbiome field.
If successful, our proposal will identify new disease risk factors and risk prediction tools based on the microbiome, paving the way towards using the microbiome in early disease detection and prevention.

Identifying risk factors for diseases that can be prevented or delayed by early intervention is of major importance, and numerous genetic, lifestyle, anthropometric and clinical risk factors were found for many different diseases. Another source of potentially pertinent disease risk factors is the human microbiome - the collective genome of trillions of bacteria, viruses, fungi, and parasites that reside in the human gut. However, very few microbiome disease markers were found to date.
Here, we aim to develop risk prediction tools based on the human microbiome that predict the likelihood of an individual to develop a particular condition or disease within 5-10 years. We will use a cohort of >2200 individuals that my group previously assembled, for whom we have clinical profiles, gut microbiome data, and banked blood and stool samples. We will invite people 5-10 years after their initial recruitment time, profile disease status and blood markers, and develop algorithms for predicting 5-10 year onset of Type 2 diabetes, cardiovascular disease, and obesity, using microbiome data from recruitment time.
To increase the likelihood of finding microbiome markers predictive of disease onset, we will develop novel experimental and computational methods for in-depth characterization of microbial gene function, the metabolites produced by the microbiome, the underexplored fungal microbiome members, and the interactions between the gut microbiota and the host adaptive immune system. We will then apply these methods to >2200 banked samples from cohort recruitment time and use the resulting data in devising our microbiome-based risk prediction tools. In themselves, these novel assays and their application to >2200 samples should greatly advance the microbiome field.
If successful, our proposal will identify new disease risk factors and risk prediction tools based on the microbiome, paving the way towards using the microbiome in early disease detection and prevention.

SummaryIn mammals, transcriptional control of many genes relies on cis-regulatory elements such as enhancers, which are often located tens to hundreds of kilobases away from their cognate promoters. Functional interactions between distal regulatory elements and target promoters require mutual physical proximity, which is linked to the three-dimensional structure of the chromatin fiber. Chromosome conformation capture studies revealed that chromosomes are partitioned into Topologically Associating Domains (TADs), sub-megabase domains of preferential physical interactions of the chromatin fiber. Genetic evidence showed that TAD boundaries restrict the genomic range of enhancer-promoter communication, and that interactions between regulatory sequences within TADs are further fine-tuned by smaller-scale structures. However, the mechanistic details of how physical interactions translate into transcriptional outputs are totally unknown. Here we propose to explore the biophysical mechanisms that link chromosome conformation and long-range transcriptional regulation using molecular biology, genetic engineering, single-cell experiments and physical modeling. We will measure chromosomal interactions in single cells and in time using a novel method that relies on an enzymatic process in vivo. Genetic engineering will be used to establish a cell system that allows quantitative measurement of how enhancer-promoter interactions relate to transcription at the population and single-cell levels, and to test the effects of perturbations without confounding effects. Finally, we will develop physical models of promoter operation in the presence of distal enhancers, which will be used to interpret the experimental data and formulate new testable predictions. With this integrated approach we aim at providing an entirely new layer of description of the general principles underlying transcriptional control, which could establish new paradigms for research in epigenetics and gene regulation.

In mammals, transcriptional control of many genes relies on cis-regulatory elements such as enhancers, which are often located tens to hundreds of kilobases away from their cognate promoters. Functional interactions between distal regulatory elements and target promoters require mutual physical proximity, which is linked to the three-dimensional structure of the chromatin fiber. Chromosome conformation capture studies revealed that chromosomes are partitioned into Topologically Associating Domains (TADs), sub-megabase domains of preferential physical interactions of the chromatin fiber. Genetic evidence showed that TAD boundaries restrict the genomic range of enhancer-promoter communication, and that interactions between regulatory sequences within TADs are further fine-tuned by smaller-scale structures. However, the mechanistic details of how physical interactions translate into transcriptional outputs are totally unknown. Here we propose to explore the biophysical mechanisms that link chromosome conformation and long-range transcriptional regulation using molecular biology, genetic engineering, single-cell experiments and physical modeling. We will measure chromosomal interactions in single cells and in time using a novel method that relies on an enzymatic process in vivo. Genetic engineering will be used to establish a cell system that allows quantitative measurement of how enhancer-promoter interactions relate to transcription at the population and single-cell levels, and to test the effects of perturbations without confounding effects. Finally, we will develop physical models of promoter operation in the presence of distal enhancers, which will be used to interpret the experimental data and formulate new testable predictions. With this integrated approach we aim at providing an entirely new layer of description of the general principles underlying transcriptional control, which could establish new paradigms for research in epigenetics and gene regulation.

Max ERC Funding

1 500 000 €

Duration

Start date: 2018-01-01, End date: 2022-12-31

Project acronymCellKarma

ProjectDissecting the regulatory logic of cell fate reprogramming through integrative and single cell genomics

Researcher (PI)Davide CACCHIARELLI

Host Institution (HI)FONDAZIONE TELETHON

Call DetailsStarting Grant (StG), LS2, ERC-2017-STG

SummaryThe concept that any cell type, upon delivery of the right “cocktail” of transcription factors, can acquire an identity that otherwise it would never achieve, revolutionized the way we approach the study of developmental biology. In light of this, the discovery of induced pluripotent stem cells (IPSCs) and cell fate conversion approaches stimulated new research directions into human regenerative biology. However, the chance to successfully develop patient-tailored therapies is still very limited because reprogramming technologies are applied without a comprehensive understanding of the molecular processes involved.
Here, I propose a multifaceted approach that combines a wide range of cutting-edge integrative genomic strategies to significantly advance our understanding of the regulatory logic driving cell fate decisions during human reprogramming to pluripotency.
To this end, I will utilize single cell transcriptomics to isolate reprogramming intermediates, reconstruct their lineage relationships and define transcriptional regulators responsible for the observed transitions (AIM 1). Then, I will dissect the rules by which transcription factors modulate the activity of promoters and enhancer regions during reprogramming transitions, by applying synthetic biology and genome editing approaches (AIM 2). Then, I will adopt an alternative approach to identify reprogramming modulators by the analysis of reprogramming-induced mutagenesis events (AIM 3). Finally, I will explore my findings in multiple primary reprogramming approaches to pluripotency, with the ultimate goal of improving the quality of IPSC derivation (Aim 4).
In summary, this project will expose novel determinants and yet unidentified molecular barriers of reprogramming to pluripotency and will be essential to unlock the full potential of reprogramming technologies for shaping cellular identity in vitro and to address pressing challenges of regenerative medicine.

The concept that any cell type, upon delivery of the right “cocktail” of transcription factors, can acquire an identity that otherwise it would never achieve, revolutionized the way we approach the study of developmental biology. In light of this, the discovery of induced pluripotent stem cells (IPSCs) and cell fate conversion approaches stimulated new research directions into human regenerative biology. However, the chance to successfully develop patient-tailored therapies is still very limited because reprogramming technologies are applied without a comprehensive understanding of the molecular processes involved.
Here, I propose a multifaceted approach that combines a wide range of cutting-edge integrative genomic strategies to significantly advance our understanding of the regulatory logic driving cell fate decisions during human reprogramming to pluripotency.
To this end, I will utilize single cell transcriptomics to isolate reprogramming intermediates, reconstruct their lineage relationships and define transcriptional regulators responsible for the observed transitions (AIM 1). Then, I will dissect the rules by which transcription factors modulate the activity of promoters and enhancer regions during reprogramming transitions, by applying synthetic biology and genome editing approaches (AIM 2). Then, I will adopt an alternative approach to identify reprogramming modulators by the analysis of reprogramming-induced mutagenesis events (AIM 3). Finally, I will explore my findings in multiple primary reprogramming approaches to pluripotency, with the ultimate goal of improving the quality of IPSC derivation (Aim 4).
In summary, this project will expose novel determinants and yet unidentified molecular barriers of reprogramming to pluripotency and will be essential to unlock the full potential of reprogramming technologies for shaping cellular identity in vitro and to address pressing challenges of regenerative medicine.

Max ERC Funding

1 497 250 €

Duration

Start date: 2018-03-01, End date: 2023-02-28

Project acronymCENEVO

ProjectA new paradigm for centromere biology:Evolution and mechanism of CenH3-independent chromosome segregation in holocentric insects

Researcher (PI)Ines DRINNENBERG

Host Institution (HI)INSTITUT CURIE

Call DetailsStarting Grant (StG), LS2, ERC-2017-STG

SummaryFaithful chromosome segregation in all eukaryotes relies on centromeres, the chromosomal sites that recruit kinetochore proteins and mediate spindle attachment during cell division. Fundamental to centromere function is a histone H3 variant, CenH3, that initiates kinetochore assembly on centromeric DNA. CenH3 is conserved throughout most eukaryotes; its deletion is lethal in all organisms tested. These findings established the paradigm that CenH3 is an absolute requirement for centromere function. My recent findings undermined this paradigm of CenH3 essentiality. I showed that CenH3 was lost independently in four lineages of insects. These losses are concomitant with dramatic changes in their centromeric architecture, in which each lineage independently transitioned from monocentromeres (where microtubules attach to a single chromosomal region) to holocentromeres (where microtubules attach along the entire length of the chromosome). Here, I aim to characterize this unique CenH3-deficient chromosome segregation pathway. Using proteomic and genomic approaches in lepidopteran cell lines, I will determine the mechanism of CenH3-independent kinetochore assembly that led to the establishment of their holocentric architecture. Using comparative genomic approaches, I will determine whether this kinetochore assembly pathway has recurrently evolved over the course of 400 million years of evolution and its impact on the chromosome segregation machinery.
My discovery of CenH3 loss in holocentric insects establishes a new class of centromeres. My research will reveal how CenH3 that is essential in most other eukaryotes, could have become dispensable in holocentric insects. Since the evolution of this CenH3-independent chromosome segregation pathway is associated with the independent rises of holocentric architectures, my research will also provide the first insights into the transition from a monocentromere to a holocentromere.

Faithful chromosome segregation in all eukaryotes relies on centromeres, the chromosomal sites that recruit kinetochore proteins and mediate spindle attachment during cell division. Fundamental to centromere function is a histone H3 variant, CenH3, that initiates kinetochore assembly on centromeric DNA. CenH3 is conserved throughout most eukaryotes; its deletion is lethal in all organisms tested. These findings established the paradigm that CenH3 is an absolute requirement for centromere function. My recent findings undermined this paradigm of CenH3 essentiality. I showed that CenH3 was lost independently in four lineages of insects. These losses are concomitant with dramatic changes in their centromeric architecture, in which each lineage independently transitioned from monocentromeres (where microtubules attach to a single chromosomal region) to holocentromeres (where microtubules attach along the entire length of the chromosome). Here, I aim to characterize this unique CenH3-deficient chromosome segregation pathway. Using proteomic and genomic approaches in lepidopteran cell lines, I will determine the mechanism of CenH3-independent kinetochore assembly that led to the establishment of their holocentric architecture. Using comparative genomic approaches, I will determine whether this kinetochore assembly pathway has recurrently evolved over the course of 400 million years of evolution and its impact on the chromosome segregation machinery.
My discovery of CenH3 loss in holocentric insects establishes a new class of centromeres. My research will reveal how CenH3 that is essential in most other eukaryotes, could have become dispensable in holocentric insects. Since the evolution of this CenH3-independent chromosome segregation pathway is associated with the independent rises of holocentric architectures, my research will also provide the first insights into the transition from a monocentromere to a holocentromere.

Max ERC Funding

1 497 500 €

Duration

Start date: 2018-04-01, End date: 2023-03-31

Project acronymCharFL

ProjectCharacterizing the fitness landscape on population and global scales

Researcher (PI)Fyodor Kondrashov

Host Institution (HI)INSTITUTE OF SCIENCE AND TECHNOLOGYAUSTRIA

Call DetailsConsolidator Grant (CoG), LS2, ERC-2017-COG

SummaryThe fitness landscape, the representation of how the genotype manifests at the phenotypic (fitness) levels, may be among the most useful concepts in biology with impact on diverse fields, including quantitative genetics, emergence of pathogen resistance, synthetic biology and protein engineering. While progress in characterizing fitness landscapes has been made, three directions of research in the field remain virtually unexplored: the nature of the genotype to phenotype of standing variation (variation found in a natural population), the shape of the fitness landscape encompassing many genotypes and the modelling of complex genetic interactions in protein sequences.
The current proposal is designed to advance the study of fitness landscapes in these three directions using large-scale genomic experiments and experimental data from a model protein and theoretical work. The study of the fitness landscape of standing variation is aimed at the resolution of an outstanding question in quantitative genetics: the extent to which epistasis, non-additive genetic interactions, is shaping the phenotype. The second aim of characterizing the global fitness landscape will give us an understanding of how evolution proceeds along long evolutionary timescales, which can be directly applied to protein engineering and synthetic biology for the design of novel phenotypes. Finally, the third aim of modelling complex interactions will improve our ability to predict phenotypes from genotypes, such as the prediction of human disease mutations. In summary, the proposed study presents an opportunity to provide a unifying understanding of how phenotypes are shaped through genetic interactions. The consolidation of our empirical and theoretical work on different scales of the genotype to phenotype relationship will provide empirical data and novel context for several fields of biology.

The fitness landscape, the representation of how the genotype manifests at the phenotypic (fitness) levels, may be among the most useful concepts in biology with impact on diverse fields, including quantitative genetics, emergence of pathogen resistance, synthetic biology and protein engineering. While progress in characterizing fitness landscapes has been made, three directions of research in the field remain virtually unexplored: the nature of the genotype to phenotype of standing variation (variation found in a natural population), the shape of the fitness landscape encompassing many genotypes and the modelling of complex genetic interactions in protein sequences.
The current proposal is designed to advance the study of fitness landscapes in these three directions using large-scale genomic experiments and experimental data from a model protein and theoretical work. The study of the fitness landscape of standing variation is aimed at the resolution of an outstanding question in quantitative genetics: the extent to which epistasis, non-additive genetic interactions, is shaping the phenotype. The second aim of characterizing the global fitness landscape will give us an understanding of how evolution proceeds along long evolutionary timescales, which can be directly applied to protein engineering and synthetic biology for the design of novel phenotypes. Finally, the third aim of modelling complex interactions will improve our ability to predict phenotypes from genotypes, such as the prediction of human disease mutations. In summary, the proposed study presents an opportunity to provide a unifying understanding of how phenotypes are shaped through genetic interactions. The consolidation of our empirical and theoretical work on different scales of the genotype to phenotype relationship will provide empirical data and novel context for several fields of biology.

Max ERC Funding

1 998 280 €

Duration

Start date: 2019-01-01, End date: 2023-12-31

Project acronymCHROMATADS

ProjectChromatin Packing and Architectural Proteins in Plants

Researcher (PI)Chang LIU

Host Institution (HI)EBERHARD KARLS UNIVERSITAET TUEBINGEN

Call DetailsStarting Grant (StG), LS2, ERC-2017-STG

SummaryThe three-dimensional organization of the genome, which strikingly correlates with gene activity, is critical for many cellular processes. The evolution of molecular techniques has allowed us to unveil chromatin structure at an unprecedented resolution. The most intriguing chromatin structures observed in animals are TADs (Topologically Associating Domains), which represent the functional and structural chromatin domains demarcating the genome. Structural proteins such as insulators proteins, on the other hand, have been shown to play crucial roles in mediating the formation of TADs. However, major structural factors relevant to chromatin structure are still waiting to be discovered in land plants. My preliminary work shows that TADs are widely distributed across the rice genome, and motif sequence analysis suggests the enrichment of plant-specific transcription factors at TAD boundaries, which jointly give rise to an exciting hypothesis that these proteins might be the long-sought-after insulators in land plants. By using various state-of-the-art molecular and computational tools, this timely project aims to fill a huge gap in plant functional genomics and substantially advance our understanding of three-dimensional chromatin structure. This project consists four major aims, which collectively will uncover the identities of plant insulator proteins and generate insights into the dynamics of structural chromatin domains during stress adaptation. Aim 1 will identify and characterize the stability and plasticity of functional chromatin domains in the rice genome during temperature stress adaptation. Aim 2 will identify insulator elements and other structural features of chromatin packing in the Marchantia polymorpha genome from a structural genomics approach. Aim 3 will establish the role of candidate proteins as plant insulators. Lastly, Aim 4 will generate functional insights into the molecular mechanism by which plant insulators shape the three-dimensional genome.

The three-dimensional organization of the genome, which strikingly correlates with gene activity, is critical for many cellular processes. The evolution of molecular techniques has allowed us to unveil chromatin structure at an unprecedented resolution. The most intriguing chromatin structures observed in animals are TADs (Topologically Associating Domains), which represent the functional and structural chromatin domains demarcating the genome. Structural proteins such as insulators proteins, on the other hand, have been shown to play crucial roles in mediating the formation of TADs. However, major structural factors relevant to chromatin structure are still waiting to be discovered in land plants. My preliminary work shows that TADs are widely distributed across the rice genome, and motif sequence analysis suggests the enrichment of plant-specific transcription factors at TAD boundaries, which jointly give rise to an exciting hypothesis that these proteins might be the long-sought-after insulators in land plants. By using various state-of-the-art molecular and computational tools, this timely project aims to fill a huge gap in plant functional genomics and substantially advance our understanding of three-dimensional chromatin structure. This project consists four major aims, which collectively will uncover the identities of plant insulator proteins and generate insights into the dynamics of structural chromatin domains during stress adaptation. Aim 1 will identify and characterize the stability and plasticity of functional chromatin domains in the rice genome during temperature stress adaptation. Aim 2 will identify insulator elements and other structural features of chromatin packing in the Marchantia polymorpha genome from a structural genomics approach. Aim 3 will establish the role of candidate proteins as plant insulators. Lastly, Aim 4 will generate functional insights into the molecular mechanism by which plant insulators shape the three-dimensional genome.

Max ERC Funding

1 498 216 €

Duration

Start date: 2018-01-01, End date: 2022-12-31

Project acronymCHROMTOPOLOGY

ProjectUnderstanding and manipulating the dynamics of chromosome topologies in transcriptional control

SummaryTranscriptional regulation of genes in eukaryotic cells requires a complex and highly regulated interplay of chromatin environment, epigenetic status of target sequences and several different transcription factors. Eukaryotic genomes are tightly packaged within nuclei, yet must be accessible for transcription, replication and repair. A striking correlation exists between chromatin topology and underlying gene activity. According to the textbook view, chromatin loops bring genes into direct contact with distal regulatory elements, such as enhancers. Moreover, we and others have shown that genomes are organized into discretely folded megabase-sized regions, denoted as topologically associated domains (TADs), which seem to correlate well with transcription activity and histone modifications. However, it is unknown whether chromosome folding is a cause or consequence of underlying gene function.
To better understand the role of genome organization in transcription regulation, I will address the following questions:
(i) How are chromatin configurations altered during transcriptional changes accompanying development?
(ii) What are the real-time kinetics and cell-to-cell variabilities of chromatin interactions and TAD architectures?
(iii) Can chromatin loops be engineered de novo, and do they influence gene expression?
(iv) What genetic elements and trans-acting factors are required to organize TADs?
To address these fundamental questions, I will use a combination of novel technologies and approaches, such as Hi-C, CRISPR knock-ins, ANCHOR tagging of DNA loci, high- and super-resolution single-cell imaging, genome-wide screens and optogenetics, in order to both study and engineer chromatin architectures.
These studies will give groundbreaking insight into if and how chromatin topology regulates transcription. Thus, I anticipate that the results of this project will have a major impact on the field and will lead to a new paradigm for metazoan transcription control.

Transcriptional regulation of genes in eukaryotic cells requires a complex and highly regulated interplay of chromatin environment, epigenetic status of target sequences and several different transcription factors. Eukaryotic genomes are tightly packaged within nuclei, yet must be accessible for transcription, replication and repair. A striking correlation exists between chromatin topology and underlying gene activity. According to the textbook view, chromatin loops bring genes into direct contact with distal regulatory elements, such as enhancers. Moreover, we and others have shown that genomes are organized into discretely folded megabase-sized regions, denoted as topologically associated domains (TADs), which seem to correlate well with transcription activity and histone modifications. However, it is unknown whether chromosome folding is a cause or consequence of underlying gene function.
To better understand the role of genome organization in transcription regulation, I will address the following questions:
(i) How are chromatin configurations altered during transcriptional changes accompanying development?
(ii) What are the real-time kinetics and cell-to-cell variabilities of chromatin interactions and TAD architectures?
(iii) Can chromatin loops be engineered de novo, and do they influence gene expression?
(iv) What genetic elements and trans-acting factors are required to organize TADs?
To address these fundamental questions, I will use a combination of novel technologies and approaches, such as Hi-C, CRISPR knock-ins, ANCHOR tagging of DNA loci, high- and super-resolution single-cell imaging, genome-wide screens and optogenetics, in order to both study and engineer chromatin architectures.
These studies will give groundbreaking insight into if and how chromatin topology regulates transcription. Thus, I anticipate that the results of this project will have a major impact on the field and will lead to a new paradigm for metazoan transcription control.

SummaryThe 3D organization of chromosomes within the nucleus is of great importance to control gene expression. The cohesin complex plays a key role in such higher-order chromosome organization by looping together regulatory elements in cis. How these often megabase-sized looped structures are formed is one of the main open questions in chromosome biology. Cohesin is a ring-shaped complex that can entrap DNA inside its lumen. However, cohesin’s default behaviour is that it only transiently entraps and then releases DNA. Our recent findings indicate that chromosomes are structured through the processive enlargement of chromatin loops, and that the duration with which cohesin embraces DNA determines the degree to which loops are enlarged. The goal of this proposal is two-fold. First, we plan to investigate the mechanism by which chromatin loops are formed, and secondly we wish to dissect how looped structures are maintained. We will use a multi-disciplinary approach that includes refined genetic screens in haploid human cells, chromosome conformation capture techniques, the tracing in vivo of cohesin on individual DNA molecules, and visualization of chromosome organization by super-resolution imaging. With unbiased genetic screens, we have identified chromatin regulators involved in the formation of chromosomal loops. We will investigate how they drive loop formation, and also whether cohesin’s own enzymatic activity plays a role in the enlargement of loops. We will study whether and how these factors control the movement of cohesin along individual DNA molecules, and whether chromatin loops pass through cohesin rings during their formation. Ultimately, we plan to couple cohesin’s linear trajectory along chromatin to the 3D consequences for chromosomal architecture. Together our experiments will provide vital insight into how cohesin structures chromosomes.

The 3D organization of chromosomes within the nucleus is of great importance to control gene expression. The cohesin complex plays a key role in such higher-order chromosome organization by looping together regulatory elements in cis. How these often megabase-sized looped structures are formed is one of the main open questions in chromosome biology. Cohesin is a ring-shaped complex that can entrap DNA inside its lumen. However, cohesin’s default behaviour is that it only transiently entraps and then releases DNA. Our recent findings indicate that chromosomes are structured through the processive enlargement of chromatin loops, and that the duration with which cohesin embraces DNA determines the degree to which loops are enlarged. The goal of this proposal is two-fold. First, we plan to investigate the mechanism by which chromatin loops are formed, and secondly we wish to dissect how looped structures are maintained. We will use a multi-disciplinary approach that includes refined genetic screens in haploid human cells, chromosome conformation capture techniques, the tracing in vivo of cohesin on individual DNA molecules, and visualization of chromosome organization by super-resolution imaging. With unbiased genetic screens, we have identified chromatin regulators involved in the formation of chromosomal loops. We will investigate how they drive loop formation, and also whether cohesin’s own enzymatic activity plays a role in the enlargement of loops. We will study whether and how these factors control the movement of cohesin along individual DNA molecules, and whether chromatin loops pass through cohesin rings during their formation. Ultimately, we plan to couple cohesin’s linear trajectory along chromatin to the 3D consequences for chromosomal architecture. Together our experiments will provide vital insight into how cohesin structures chromosomes.

Max ERC Funding

1 998 375 €

Duration

Start date: 2018-04-01, End date: 2023-03-31

Project acronymCTCFStableGenome

ProjectCTCF control of genome stability in ageing

Researcher (PI)Duncan ODOM

Host Institution (HI)DEUTSCHES KREBSFORSCHUNGSZENTRUM HEIDELBERG

Call DetailsAdvanced Grant (AdG), LS2, ERC-2017-ADG

Summary. Genome stability is one of the most important features in maintaining tissue homeostasis throughout the human lifespan. The research presented here will dissect how the insulator protein CCCTC-binding factor (CTCF), a ubiquitous 11 zinc finger transcription factor, controls the stability of the mammalian genome during ageing.
. In Aim 1, we will elucidate how CTCF and tissue-specific master regulators maintain the functional stability of the genome during healthy ageing by developing a novel protocol to map simultaneously transcription and open chromatin in isolated hepatocyte nuclei. Using this protocol, we will explore how CTCF binding stabilizes cellular homeostasis during ageing by knocking down CTCF in vivo, both in isolation and simultaneously with knock down of liver-specific master regulators.
. In Aim 2, we will reveal the molecular mechanisms underlying CTCF binding sites as susceptibility loci for somatic mutations. We will profile the mutations in open chromatin of single nuclei immediately following acute exposure to a chemical mutagen; comparing how the pattern of mutations in CTCF bound regions changes across an allelic series of CTCF knockdown mice will reveal how CTCF binding shapes the stability of the genome towards mutations.
. These integrated strategies develop and deploy powerful, cutting-edge experimental approaches to reveal novel aspects of how CTCF binding stabilises the mammalian genome during healthy ageing as well as during mutagenesis.

. Genome stability is one of the most important features in maintaining tissue homeostasis throughout the human lifespan. The research presented here will dissect how the insulator protein CCCTC-binding factor (CTCF), a ubiquitous 11 zinc finger transcription factor, controls the stability of the mammalian genome during ageing.
. In Aim 1, we will elucidate how CTCF and tissue-specific master regulators maintain the functional stability of the genome during healthy ageing by developing a novel protocol to map simultaneously transcription and open chromatin in isolated hepatocyte nuclei. Using this protocol, we will explore how CTCF binding stabilizes cellular homeostasis during ageing by knocking down CTCF in vivo, both in isolation and simultaneously with knock down of liver-specific master regulators.
. In Aim 2, we will reveal the molecular mechanisms underlying CTCF binding sites as susceptibility loci for somatic mutations. We will profile the mutations in open chromatin of single nuclei immediately following acute exposure to a chemical mutagen; comparing how the pattern of mutations in CTCF bound regions changes across an allelic series of CTCF knockdown mice will reveal how CTCF binding shapes the stability of the genome towards mutations.
. These integrated strategies develop and deploy powerful, cutting-edge experimental approaches to reveal novel aspects of how CTCF binding stabilises the mammalian genome during healthy ageing as well as during mutagenesis.

SummaryUnderstanding how genomic information is organised and interpreted to give rise to robust patterns of gene expression is a long-standing problem in genome biology, with direct implications for development, evolution and disease. Despite recent advances in locating regulatory elements in animal genomes, there is a general lack of functional data on elements in their endogenous setting – the bulk of our current knowledge comes from reporter assays examining elements out of context, giving insights on sufficiency but not necessity. The functional requirement of very few individual enhancers, and other elements, has been assessed by deletion, with even less known about how the action of multiple elements is integrated. To understand the functional effects of genetic variants, and how they are buffered during embryogenesis, it is imperative to genetically dissect regulatory domains to uncover functional rules of genome regulation within a well-characterised animal model. Here, by combining Drosophila population genetics, developmental genetics, and novel multiplexed genomic methods we will perform the first large-scale functional dissection of cis-regulatory landscapes during embryogenesis.
Extensive resources make Drosophila a unique model organism for this task, including (a) 500 fully sequenced inbred wild isolates for population genetics, (b) over 20,000 fly strains custom-built for genome engineering & (c) a wealth of cis-regulatory information on the location of enhancers. The proposal has three Aims: 1) Use population genetics as a perturbation tool to functionally link regulatory elements to their target genes; 2) Systematically delete cis-regulatory elements to dissect their role in gene expression and genome topology; 3) Manipulate cis-regulatory domains to generate new regulatory environments for developmental genes.These Aims will provide unique functional insights, enabling us to move from correlation to causation in our understanding of genome regulation.

Understanding how genomic information is organised and interpreted to give rise to robust patterns of gene expression is a long-standing problem in genome biology, with direct implications for development, evolution and disease. Despite recent advances in locating regulatory elements in animal genomes, there is a general lack of functional data on elements in their endogenous setting – the bulk of our current knowledge comes from reporter assays examining elements out of context, giving insights on sufficiency but not necessity. The functional requirement of very few individual enhancers, and other elements, has been assessed by deletion, with even less known about how the action of multiple elements is integrated. To understand the functional effects of genetic variants, and how they are buffered during embryogenesis, it is imperative to genetically dissect regulatory domains to uncover functional rules of genome regulation within a well-characterised animal model. Here, by combining Drosophila population genetics, developmental genetics, and novel multiplexed genomic methods we will perform the first large-scale functional dissection of cis-regulatory landscapes during embryogenesis.
Extensive resources make Drosophila a unique model organism for this task, including (a) 500 fully sequenced inbred wild isolates for population genetics, (b) over 20,000 fly strains custom-built for genome engineering & (c) a wealth of cis-regulatory information on the location of enhancers. The proposal has three Aims: 1) Use population genetics as a perturbation tool to functionally link regulatory elements to their target genes; 2) Systematically delete cis-regulatory elements to dissect their role in gene expression and genome topology; 3) Manipulate cis-regulatory domains to generate new regulatory environments for developmental genes.These Aims will provide unique functional insights, enabling us to move from correlation to causation in our understanding of genome regulation.

SummaryCancers develop in very heterogeneous tissue environments. They depend on the tumor microenvironment (TME) for sustained growth, metastasis, and therapy resistance. Stromal cells are genetically stable and they have less likelihood to develop resistance than cancer cells. Therefore, targeting the TME represents an attractive approach for treating cancer. In order to develop new therapeutic strategies to reprogram the TME and inhibit tumor growth and resistance, it is essential to understand in detail the molecular mechanisms of the interactions between cancer and stromal cell populations. However, current methods to study these interactions require complete dissociation of the tumor, exposing the cells to severe stress and affecting dramatically gene expression patterns. Here, I propose to use Dual Ribosome Profiling (DualRP), a system that I recently developed, to study cell interactions in the TME. DualRP is an approach that allows not only simultaneous analysis of gene expression in two interacting cell populations in vivo, but also is able to uncover metabolic limitations in tumors. I aim to apply DualRP to mouse xenograft models where cancer cells interact with non-transformed fibroblasts and I’ll explore the combined response of both populations to cancer therapy. Moreover, I’ll utilize mouse genetic models tailored for DualRP to study cancer cell and macrophages/endothelial cells interactions. I will employ a combination of mouse genetic models, biochemical tools, deep sequencing, and bioinformatics. These studies will provide insight into how gene expression and metabolic programs define the interaction between cancer and stromal cells to promote tumor growth and metastasis, identify potential targets for therapeutic intervention, and provide maps of cell interactions in vivo. Therefore, this research has the potential to significantly advance our understanding of the molecular and metabolic mechanisms underlying the complex cell interactions in the TME.

Cancers develop in very heterogeneous tissue environments. They depend on the tumor microenvironment (TME) for sustained growth, metastasis, and therapy resistance. Stromal cells are genetically stable and they have less likelihood to develop resistance than cancer cells. Therefore, targeting the TME represents an attractive approach for treating cancer. In order to develop new therapeutic strategies to reprogram the TME and inhibit tumor growth and resistance, it is essential to understand in detail the molecular mechanisms of the interactions between cancer and stromal cell populations. However, current methods to study these interactions require complete dissociation of the tumor, exposing the cells to severe stress and affecting dramatically gene expression patterns. Here, I propose to use Dual Ribosome Profiling (DualRP), a system that I recently developed, to study cell interactions in the TME. DualRP is an approach that allows not only simultaneous analysis of gene expression in two interacting cell populations in vivo, but also is able to uncover metabolic limitations in tumors. I aim to apply DualRP to mouse xenograft models where cancer cells interact with non-transformed fibroblasts and I’ll explore the combined response of both populations to cancer therapy. Moreover, I’ll utilize mouse genetic models tailored for DualRP to study cancer cell and macrophages/endothelial cells interactions. I will employ a combination of mouse genetic models, biochemical tools, deep sequencing, and bioinformatics. These studies will provide insight into how gene expression and metabolic programs define the interaction between cancer and stromal cells to promote tumor growth and metastasis, identify potential targets for therapeutic intervention, and provide maps of cell interactions in vivo. Therefore, this research has the potential to significantly advance our understanding of the molecular and metabolic mechanisms underlying the complex cell interactions in the TME.

SummaryIn eukaryotes, the complex regulation of temporal- and tissue-specific gene expression is controlled by the binding of transcription factors to enhancers, which in turn interact with the promoter of their target gene(s) via the formation of a chromatin loop. Despite their importance, the properties governing enhancer function and enhancer-promoter loops in the context of the three-dimensional organisation of the genome are still poorly understood.
My recent work suggests that (i) developmental genes are often regulated by multiple enhancers, sometimes located at great linear distances, (ii) the spatio-temporal activity of a large fraction of those enhancers remains unknown, (iii) enhancer-promoter interactions are usually established before the target gene is expressed and are largely stable during embryogenesis, and (iv) stable interactions seem to be associated with the presence of paused RNA Polymerase II at the promoter before gene activation.
Building upon these results, we propose to advance to the next level in the dissection of enhancer-promoter interaction functionality in the context of Drosophila embryogenesis. Specifically, we will address three important questions: (i) What determines the specificity of promoter-enhancer interactions in a complex genome? (ii) Are enhancer-promoter interactions tissue-specific, and what are the drivers of this specificity? (iii) Are all enhancer-promoter interactions functional, and how does the activity of an enhancer relate to the expression of the gene it interacts with?
To this end, my group will apply an interdisciplinary approach, combining state-of-the-art methods in genetics and genomics, including novel single-cell techniques, using Drosophila embryogenesis as a model system. Our results will provide a unique view of the functionality of enhancer-promoter interactions in a developing embryo, a significant step towards understanding the link between chromatin organisation and transcription regulation.

In eukaryotes, the complex regulation of temporal- and tissue-specific gene expression is controlled by the binding of transcription factors to enhancers, which in turn interact with the promoter of their target gene(s) via the formation of a chromatin loop. Despite their importance, the properties governing enhancer function and enhancer-promoter loops in the context of the three-dimensional organisation of the genome are still poorly understood.
My recent work suggests that (i) developmental genes are often regulated by multiple enhancers, sometimes located at great linear distances, (ii) the spatio-temporal activity of a large fraction of those enhancers remains unknown, (iii) enhancer-promoter interactions are usually established before the target gene is expressed and are largely stable during embryogenesis, and (iv) stable interactions seem to be associated with the presence of paused RNA Polymerase II at the promoter before gene activation.
Building upon these results, we propose to advance to the next level in the dissection of enhancer-promoter interaction functionality in the context of Drosophila embryogenesis. Specifically, we will address three important questions: (i) What determines the specificity of promoter-enhancer interactions in a complex genome? (ii) Are enhancer-promoter interactions tissue-specific, and what are the drivers of this specificity? (iii) Are all enhancer-promoter interactions functional, and how does the activity of an enhancer relate to the expression of the gene it interacts with?
To this end, my group will apply an interdisciplinary approach, combining state-of-the-art methods in genetics and genomics, including novel single-cell techniques, using Drosophila embryogenesis as a model system. Our results will provide a unique view of the functionality of enhancer-promoter interactions in a developing embryo, a significant step towards understanding the link between chromatin organisation and transcription regulation.

Max ERC Funding

1 770 375 €

Duration

Start date: 2018-05-01, End date: 2023-04-30

Project acronymEpigenomeProgramming

ProjectAn experimental and bioinformatic toolbox for functional epigenomics and its application to epigenetically making and breaking a cancer cell

SummaryEpigenetic alterations can be detected in all cancers and in essentially every patient. Despite their prevalence, the concrete functional roles of these alterations are not well understood, for two reasons: First, cancer samples tend to carry many correlated epigenetic alterations, making it difficult to statistically distinguish relevant driver events from those that co-occur for other reasons. Second, we lack tools for targeted epigenome editing that could be used to validate biological function in perturbation and rescue experiments.
The proposed project strives to overcome these limitations through experimental and bioinformatic methods development, with the ambition of making and breaking cancer cells in vitro by introducing defined sets of epigenetic alterations. We will focus on leukemia as our “model cancer” (given its low mutation rate, frequent defects in epigenetic regulators, and availability of excellent functional assays), but the concepts and methods are general. In Aim 1, we will generate epigenome profiles for a human knockout cell collection comprising 100 epigenetic regulators and use the data to functionally annotate thousands of epigenetic alterations observed in large cancer datasets. In Aim 2, we will develop an experimental toolbox for epigenome programming using epigenetic drugs, CRISPR-assisted recruitment of epigenetic modifiers for locus-specific editing, and cell-derived guide RNA libraries for epigenome copying. Finally, in Aim 3 we will explore epigenome programming (methods from Aim 2) of candidate driver events (predictions from Aim 1) with the ultimate goal of converting cancer cells into non-cancer cells and vice versa.
In summary, this project will establish a broadly applicable methodology and toolbox for dissecting the functional roles of epigenetic alterations in cancer. Moreover, successful creation of a cancer that is driven purely by epigenetic alterations could challenge our understanding of cancer as a genetic disease.

Epigenetic alterations can be detected in all cancers and in essentially every patient. Despite their prevalence, the concrete functional roles of these alterations are not well understood, for two reasons: First, cancer samples tend to carry many correlated epigenetic alterations, making it difficult to statistically distinguish relevant driver events from those that co-occur for other reasons. Second, we lack tools for targeted epigenome editing that could be used to validate biological function in perturbation and rescue experiments.
The proposed project strives to overcome these limitations through experimental and bioinformatic methods development, with the ambition of making and breaking cancer cells in vitro by introducing defined sets of epigenetic alterations. We will focus on leukemia as our “model cancer” (given its low mutation rate, frequent defects in epigenetic regulators, and availability of excellent functional assays), but the concepts and methods are general. In Aim 1, we will generate epigenome profiles for a human knockout cell collection comprising 100 epigenetic regulators and use the data to functionally annotate thousands of epigenetic alterations observed in large cancer datasets. In Aim 2, we will develop an experimental toolbox for epigenome programming using epigenetic drugs, CRISPR-assisted recruitment of epigenetic modifiers for locus-specific editing, and cell-derived guide RNA libraries for epigenome copying. Finally, in Aim 3 we will explore epigenome programming (methods from Aim 2) of candidate driver events (predictions from Aim 1) with the ultimate goal of converting cancer cells into non-cancer cells and vice versa.
In summary, this project will establish a broadly applicable methodology and toolbox for dissecting the functional roles of epigenetic alterations in cancer. Moreover, successful creation of a cancer that is driven purely by epigenetic alterations could challenge our understanding of cancer as a genetic disease.

SummaryPhenotypic variation arises from the heritable acquisition of cell-type specific gene-expression programs. Key in understanding cellular specification is to elucidate the epigenetic mechanism that underlies transcriptional heterogeneity. Thus a central question in biology is how cell-to-cell variability in the epigenome contributes to the emergence of phenotypic differences. However, current techniques to profile the epigenome require populations of cells and consequently present ensemble averages of the underlying biology. Therefore, to grasp the molecular concept behind the cellular acquisition of heritable traits it is essential to develop techniques to profile the epigenome at the single-cell level.
The advent of single-cell genomics enabled profiling of few epigenetic features and transcriptomics in single cells; however, this toolbox is still very restricted and moreover, to directly correlate the variability in the epigenome to changes in gene-expression activity it is pivotal to device methods to obtain both measurements from the same cell. Therefore, to bridge these shortcomings in the epigenetic toolbox, we plan to develop and apply novel techniques to profile the epigenome in single cells. With this proposal we aim to (1) develop a method to map histone modifications in single cells (2) develop a method to map chromatin organization in single cells (3) develop a method to obtain combined measurements of the epigenome and the transcriptome of the same cell (4) apply these and previously developed single-cell methods, to different biological systems to study how the epigenome contributes to lineage specification. Collectively, the goal of this proposal is to develop a comprehensive single-cell toolbox to take the field to the next (epigenomic) level and to work towards elucidating the molecular mechanism behind cellular specification.

Phenotypic variation arises from the heritable acquisition of cell-type specific gene-expression programs. Key in understanding cellular specification is to elucidate the epigenetic mechanism that underlies transcriptional heterogeneity. Thus a central question in biology is how cell-to-cell variability in the epigenome contributes to the emergence of phenotypic differences. However, current techniques to profile the epigenome require populations of cells and consequently present ensemble averages of the underlying biology. Therefore, to grasp the molecular concept behind the cellular acquisition of heritable traits it is essential to develop techniques to profile the epigenome at the single-cell level.
The advent of single-cell genomics enabled profiling of few epigenetic features and transcriptomics in single cells; however, this toolbox is still very restricted and moreover, to directly correlate the variability in the epigenome to changes in gene-expression activity it is pivotal to device methods to obtain both measurements from the same cell. Therefore, to bridge these shortcomings in the epigenetic toolbox, we plan to develop and apply novel techniques to profile the epigenome in single cells. With this proposal we aim to (1) develop a method to map histone modifications in single cells (2) develop a method to map chromatin organization in single cells (3) develop a method to obtain combined measurements of the epigenome and the transcriptome of the same cell (4) apply these and previously developed single-cell methods, to different biological systems to study how the epigenome contributes to lineage specification. Collectively, the goal of this proposal is to develop a comprehensive single-cell toolbox to take the field to the next (epigenomic) level and to work towards elucidating the molecular mechanism behind cellular specification.

Max ERC Funding

1 500 000 €

Duration

Start date: 2016-04-01, End date: 2021-03-31

Project acronymExtinction Genomics

ProjectExploring and exploiting the potential of extinct genome sequencing

Researcher (PI)Marcus Thomas Pius Gilbert

Host Institution (HI)KOBENHAVNS UNIVERSITET

Call DetailsConsolidator Grant (CoG), LS2, ERC-2015-CoG

SummaryPalaeogenomics is the nascent discipline concerned with sequencing and analysis of genome-scale information from historic, ancient, and even extinct samples. While once inconceivable due to the challenges of DNA damage, contamination, and the technical limitations of PCR-based Sanger sequencing, following the dawn of the second-generation sequencing revolution, it has rapidly become a reality. Indeed, so much so, that popular perception has moved away from if extinct species’ genomes can be sequenced, to when it will happen - and even, when will the first extinct animals be regenerated. Unfortunately this view is naïve, and does not account for the financial and technical challenges that face such attempts. I propose an exploration of exactly what the limits on genome reconstruction from extinct or otherwise historic/ancient material are. This will be achieved through new laboratory and bioinformatic developments aimed at decreasing the cost, while concomitantly increasing the quality of genome reconstruction from poor quality materials. In doing so I aim to build a scientifically-grounded framework against which the possibilities and limitations of extinct genome reconstruction can be assessed. Subsequently genomic information will be generated from a range of extinct and near-extinct avian and mammalian species, in order to showcase the potential of reconstructed genomes across research questions spanning at least three different streams of research: De-extinction, Evolutionary Genomics, and Conservation Genomics. Ultimately, achievement of these goals requires formation of a dedicated, closely knit team, focusing on both the methodological challenges as well as their bigger picture application to high-risk high-gain ventures. With ERC funding this can become a reality, and enable palaeogenomics to be pushed to the limits possible under modern technology.

Palaeogenomics is the nascent discipline concerned with sequencing and analysis of genome-scale information from historic, ancient, and even extinct samples. While once inconceivable due to the challenges of DNA damage, contamination, and the technical limitations of PCR-based Sanger sequencing, following the dawn of the second-generation sequencing revolution, it has rapidly become a reality. Indeed, so much so, that popular perception has moved away from if extinct species’ genomes can be sequenced, to when it will happen - and even, when will the first extinct animals be regenerated. Unfortunately this view is naïve, and does not account for the financial and technical challenges that face such attempts. I propose an exploration of exactly what the limits on genome reconstruction from extinct or otherwise historic/ancient material are. This will be achieved through new laboratory and bioinformatic developments aimed at decreasing the cost, while concomitantly increasing the quality of genome reconstruction from poor quality materials. In doing so I aim to build a scientifically-grounded framework against which the possibilities and limitations of extinct genome reconstruction can be assessed. Subsequently genomic information will be generated from a range of extinct and near-extinct avian and mammalian species, in order to showcase the potential of reconstructed genomes across research questions spanning at least three different streams of research: De-extinction, Evolutionary Genomics, and Conservation Genomics. Ultimately, achievement of these goals requires formation of a dedicated, closely knit team, focusing on both the methodological challenges as well as their bigger picture application to high-risk high-gain ventures. With ERC funding this can become a reality, and enable palaeogenomics to be pushed to the limits possible under modern technology.

SummaryChemical exchange between cells and their environment occurs at cellular membranes, the interface where biology meets chemistry. Studying mechanisms of drug resistance, I realized that SoLute Carrier proteins (SLCs), not only represent the major class of small molecule transporters, but that they are encoded by one of the most neglected group of human genes. I identified a case where an SLC controls the activity of mTOR, suggesting that other SLCs may be involved in signalling. This formed the basis for the GameofGates project proposal. The name refers to SLCs as a metaphor for cellular gates coordinating access to resources following game rules that are largely unknown but worth learning, as the acquired knowledge could impact our understanding of cellular physiology and open avenues for innovative treatment of human diseases.
GameofGates (GoG) plans the investigation of SLC function by comprehensively and deeply charting the genetic and protein interaction landscape of SLCs in a human cell line, while monitoring fitness, drug sensitivity and metabolic state. GoG aims at functionally de-orphanize many SLCs by assessing hundreds of thousands of genetic interactions as well as thousands protein and drug interactions. I hypothesize that SLC action is linked to signalling pathways and plays an important role in integration of metabolism and cell regulation for successful homeostasis. I propose that whole circuits of SLCs may be linked to particular nutrient auxotrophy states and that knowledge of these dependencies could instruct assessment of vulnerabilities in cancer cells. In turn, these could be pharmacologically exploited using existing or future drugs. Overall, GoG should position enough pieces into functional and regulatory networks in the SLC puzzle game to facilitate future work and motivate the community to embrace investigation of SLCs as conveyers of metabolic and chemical integration of cell biology with physiology and, in a wider scope, ecology.

Chemical exchange between cells and their environment occurs at cellular membranes, the interface where biology meets chemistry. Studying mechanisms of drug resistance, I realized that SoLute Carrier proteins (SLCs), not only represent the major class of small molecule transporters, but that they are encoded by one of the most neglected group of human genes. I identified a case where an SLC controls the activity of mTOR, suggesting that other SLCs may be involved in signalling. This formed the basis for the GameofGates project proposal. The name refers to SLCs as a metaphor for cellular gates coordinating access to resources following game rules that are largely unknown but worth learning, as the acquired knowledge could impact our understanding of cellular physiology and open avenues for innovative treatment of human diseases.
GameofGates (GoG) plans the investigation of SLC function by comprehensively and deeply charting the genetic and protein interaction landscape of SLCs in a human cell line, while monitoring fitness, drug sensitivity and metabolic state. GoG aims at functionally de-orphanize many SLCs by assessing hundreds of thousands of genetic interactions as well as thousands protein and drug interactions. I hypothesize that SLC action is linked to signalling pathways and plays an important role in integration of metabolism and cell regulation for successful homeostasis. I propose that whole circuits of SLCs may be linked to particular nutrient auxotrophy states and that knowledge of these dependencies could instruct assessment of vulnerabilities in cancer cells. In turn, these could be pharmacologically exploited using existing or future drugs. Overall, GoG should position enough pieces into functional and regulatory networks in the SLC puzzle game to facilitate future work and motivate the community to embrace investigation of SLCs as conveyers of metabolic and chemical integration of cell biology with physiology and, in a wider scope, ecology.

Max ERC Funding

2 389 782 €

Duration

Start date: 2016-10-01, End date: 2021-09-30

Project acronymGen-Epix

ProjectGenetic Determinants of the Epigenome

Researcher (PI)Adrian Peter BIRD

Host Institution (HI)THE UNIVERSITY OF EDINBURGH

Call DetailsAdvanced Grant (AdG), LS2, ERC-2015-AdG

SummaryDecoding of the genome during development and differentiation depends on sequence-specific DNA binding proteins that regulate transcription. The activity of transcription factors is constrained, however, by chromatin structure and by modification of histones and DNA, known collectively as the “epigenome”. Diseased states, particularly cancers, are often accompanied by epigenomic disturbances that contribute to aetiology, but despite much research the molecular determinants of chromatin and DNA marking remain poorly understood. A widespread view is that the epigenome responds to developmental decisions or environmental impacts that are memorised by the epigenetic machinery. Complementary to this “memory” hypothesis, there is evidence that the epigenome can directly reflect the underlying DNA sequence. We aim to explore genetic determinants of the epigenome based on our over-arching hypothesis that chromatin structure is influenced by the interaction of DNA binding proteins with short, frequent base sequence motifs. Prototypes for this scenario are proteins that bind to the two base pair sequence CpG. These proteins accumulate at CpG islands (CGIs), which are platforms for gene regulation, where they recruit multi-protein complexes that lay down epigenetic marks. By identifying and characterising novel DNA-binding proteins that sense global properties of the DNA sequence (e.g. base composition), we will address several major unanswered questions about genome regulation, including the origin of global DNA methylation patterns and the causal basis of higher order chromosome structures. Our research programme will advance genome biology and shed light on the role of epigenetic signalling in development. In particular it will explore the extent to which the epigenome is “hard-wired” by genes, with important implications for health.

Decoding of the genome during development and differentiation depends on sequence-specific DNA binding proteins that regulate transcription. The activity of transcription factors is constrained, however, by chromatin structure and by modification of histones and DNA, known collectively as the “epigenome”. Diseased states, particularly cancers, are often accompanied by epigenomic disturbances that contribute to aetiology, but despite much research the molecular determinants of chromatin and DNA marking remain poorly understood. A widespread view is that the epigenome responds to developmental decisions or environmental impacts that are memorised by the epigenetic machinery. Complementary to this “memory” hypothesis, there is evidence that the epigenome can directly reflect the underlying DNA sequence. We aim to explore genetic determinants of the epigenome based on our over-arching hypothesis that chromatin structure is influenced by the interaction of DNA binding proteins with short, frequent base sequence motifs. Prototypes for this scenario are proteins that bind to the two base pair sequence CpG. These proteins accumulate at CpG islands (CGIs), which are platforms for gene regulation, where they recruit multi-protein complexes that lay down epigenetic marks. By identifying and characterising novel DNA-binding proteins that sense global properties of the DNA sequence (e.g. base composition), we will address several major unanswered questions about genome regulation, including the origin of global DNA methylation patterns and the causal basis of higher order chromosome structures. Our research programme will advance genome biology and shed light on the role of epigenetic signalling in development. In particular it will explore the extent to which the epigenome is “hard-wired” by genes, with important implications for health.

Max ERC Funding

2 499 717 €

Duration

Start date: 2016-06-01, End date: 2021-05-31

Project acronymGeneBodyMethylation

ProjectResolving the Nuts and Bolts of Gene Body Methylation

Researcher (PI)Assaf Zemach

Host Institution (HI)TEL AVIV UNIVERSITY

Call DetailsStarting Grant (StG), LS2, ERC-2015-STG

SummaryDNA methylation, the covalent binding of a methyl group (CH3) to cytosine base, regulates the genome activity and plays a fundamental developmental role in eukaryotes. Its epigenetic characteristics of regulating transcription without changing the genetic code together with the ability to be transmitted through DNA replication allow organisms to memorize cellular events for many generations. DNA methylation is mostly known for its role in transcriptional silencing; however, its functional output is more complex and is influenced in part by its DNA context. Recent genomic studies, have found DNA methylation to be targeted inside sequences of actively transcribed genes, thus termed gene body methylation. Despite being an evolutionary conserved and a robust methylation pathway targeted to thousands of genes in animal and plant genomes, the function of gene body methylation is still poorly understood at both the molecular and functional level. Similar to the chicken and egg conundrum, because we do not know what gene body methylation does, therefore scientists could not apply its function to discover its regulators either. Gene body methylation is targeted to a very specific subset and subregions of genes, thus we strongly believe that specific factors exist and are missing simply because that no one has ever searched for them before. Hence, to make major breakthroughs in the field, our approach is to artificially generate gene-body-specific hypomethylated plants that together with customized genetic and biochemical systems will allow us to discover regulators and interpreters of gene body methylation. Using these unique genetic tools and novel molecular factors, we will be able to ultimately explore the particular biological roles of gene body methylation. These findings will fill the gap towards a full comprehension of the entire functional array of DNA methylation, and to its more precise exploitation in yielding better crops and in treating human diseases.

DNA methylation, the covalent binding of a methyl group (CH3) to cytosine base, regulates the genome activity and plays a fundamental developmental role in eukaryotes. Its epigenetic characteristics of regulating transcription without changing the genetic code together with the ability to be transmitted through DNA replication allow organisms to memorize cellular events for many generations. DNA methylation is mostly known for its role in transcriptional silencing; however, its functional output is more complex and is influenced in part by its DNA context. Recent genomic studies, have found DNA methylation to be targeted inside sequences of actively transcribed genes, thus termed gene body methylation. Despite being an evolutionary conserved and a robust methylation pathway targeted to thousands of genes in animal and plant genomes, the function of gene body methylation is still poorly understood at both the molecular and functional level. Similar to the chicken and egg conundrum, because we do not know what gene body methylation does, therefore scientists could not apply its function to discover its regulators either. Gene body methylation is targeted to a very specific subset and subregions of genes, thus we strongly believe that specific factors exist and are missing simply because that no one has ever searched for them before. Hence, to make major breakthroughs in the field, our approach is to artificially generate gene-body-specific hypomethylated plants that together with customized genetic and biochemical systems will allow us to discover regulators and interpreters of gene body methylation. Using these unique genetic tools and novel molecular factors, we will be able to ultimately explore the particular biological roles of gene body methylation. These findings will fill the gap towards a full comprehension of the entire functional array of DNA methylation, and to its more precise exploitation in yielding better crops and in treating human diseases.

Max ERC Funding

1 500 000 €

Duration

Start date: 2016-10-01, End date: 2021-09-30

Project acronymGoCADiSC

ProjectGenomics of Chromosome Architecture and Dynamics in Single Cells

Summary"The spatial architecture of mammalian interphase chromosomes, each consisting of tens of megabases of DNA, poses an intriguing topological problem and is relevant for various nuclear functions. A major challenge is that chromosome architecture exhibits substantial stochastic cell-to-cell variation. To unravel the principles of chromosome organization, new single-cell genome-wide approaches that capture the intrinsic variability are needed.
Interphase chromosomes interact extensively with relatively fixed nuclear ""landmarks"" such as the nuclear lamina and nucleoli, posing considerable restraints to the spatial organization of chromosomes. For example, about one-third of the mammalian genome interacts with the nuclear lamina. We have recently developed two complementary methods to (i) visualize and track landmark – genome interactions in living cells, and (ii) generate genome-wide maps of these interactions in single cells. These new methods offer unique opportunities to unravel chromosome architecture, taking cell-to-cell variation and dynamics into account.
Here I propose to take an integrative approach to study genome – landmark interactions in single mammalian cells. We will: (1) Extend our single-cell methods to visualize and map interactions of the genome with multiple landmarks, and with substantially enhanced genomic and temporal resolution; (2) Elucidate the dynamics and diversity of chromosome architecture in single cells, including differentiating cells; (3) Identify cis-determinants of chromosome - landmark interactions through systematic perturbation of linear chromosome organization, both by targeted mutagenesis and by a random scrambling approach; (4) elucidate the role of various proteins in the global and local control of single-cell dynamics of chromosome organization.
These tightly linked approaches will provide detailed understanding of the dynamic architecture of chromosomes in individual cells, and yield new methods and resources.
"

"The spatial architecture of mammalian interphase chromosomes, each consisting of tens of megabases of DNA, poses an intriguing topological problem and is relevant for various nuclear functions. A major challenge is that chromosome architecture exhibits substantial stochastic cell-to-cell variation. To unravel the principles of chromosome organization, new single-cell genome-wide approaches that capture the intrinsic variability are needed.
Interphase chromosomes interact extensively with relatively fixed nuclear ""landmarks"" such as the nuclear lamina and nucleoli, posing considerable restraints to the spatial organization of chromosomes. For example, about one-third of the mammalian genome interacts with the nuclear lamina. We have recently developed two complementary methods to (i) visualize and track landmark – genome interactions in living cells, and (ii) generate genome-wide maps of these interactions in single cells. These new methods offer unique opportunities to unravel chromosome architecture, taking cell-to-cell variation and dynamics into account.
Here I propose to take an integrative approach to study genome – landmark interactions in single mammalian cells. We will: (1) Extend our single-cell methods to visualize and map interactions of the genome with multiple landmarks, and with substantially enhanced genomic and temporal resolution; (2) Elucidate the dynamics and diversity of chromosome architecture in single cells, including differentiating cells; (3) Identify cis-determinants of chromosome - landmark interactions through systematic perturbation of linear chromosome organization, both by targeted mutagenesis and by a random scrambling approach; (4) elucidate the role of various proteins in the global and local control of single-cell dynamics of chromosome organization.
These tightly linked approaches will provide detailed understanding of the dynamic architecture of chromosomes in individual cells, and yield new methods and resources.
"

Max ERC Funding

2 497 125 €

Duration

Start date: 2017-03-01, End date: 2022-02-28

Project acronymGrowCELL

ProjectThe smallest of the small: determining size through cell number

Researcher (PI)Andrew JACKSON

Host Institution (HI)THE UNIVERSITY OF EDINBURGH

Call DetailsAdvanced Grant (AdG), LS2, ERC-2017-ADG

SummaryDetermination of organismal size is a fundamental biological question. Vertebrate size is established based on total cell number generated during development. Despite the 75 million-fold difference in size between the smallest and largest mammals, the mechanisms for this remain to be determined. This proposal seeks insight into how total cell number is determined in both pathological and physiological states.
Over the last decade our study of extreme growth disorders has identified 18 new human disease genes. We established these encode core components of the cell-cycle machinery, providing cellular and developmental insights into the pathophysiological mechanisms of these disorders. From our starting point of human disease, this approach also revealed novel genome instability genes informing fundamental research of basic biological processes. Still, the molecular basis for over half of individuals with microcephalic dwarfism remains unknown.
This proposal will break new ground through the comprehensive application of Whole Genome Sequencing to our patient cohort to achieve screen saturation via identification of coding and non-coding mutations. Forward-genetic genome-wide CRISPR screens in developmentally relevant cell and organoid systems will also be developed to define key cellular processes impacting human growth. Beyond these ‘discovery science’ approaches, cellular and model organism techniques will be used to define the mechanistic basis for human disease caused by mutations in core replication machinery and key epigenetic factors. To extend prior work on pathophysiological mechanisms, we aim to establish a subset of microcephalic dwarfism genes as growth regulators, and thereby further define when and how organism size is determined. These studies will link essential cellular machinery governing proliferation with human disease, identify novel genome-stability factors and may yield insights into the developmental regulation of mammalian size.

Determination of organismal size is a fundamental biological question. Vertebrate size is established based on total cell number generated during development. Despite the 75 million-fold difference in size between the smallest and largest mammals, the mechanisms for this remain to be determined. This proposal seeks insight into how total cell number is determined in both pathological and physiological states.
Over the last decade our study of extreme growth disorders has identified 18 new human disease genes. We established these encode core components of the cell-cycle machinery, providing cellular and developmental insights into the pathophysiological mechanisms of these disorders. From our starting point of human disease, this approach also revealed novel genome instability genes informing fundamental research of basic biological processes. Still, the molecular basis for over half of individuals with microcephalic dwarfism remains unknown.
This proposal will break new ground through the comprehensive application of Whole Genome Sequencing to our patient cohort to achieve screen saturation via identification of coding and non-coding mutations. Forward-genetic genome-wide CRISPR screens in developmentally relevant cell and organoid systems will also be developed to define key cellular processes impacting human growth. Beyond these ‘discovery science’ approaches, cellular and model organism techniques will be used to define the mechanistic basis for human disease caused by mutations in core replication machinery and key epigenetic factors. To extend prior work on pathophysiological mechanisms, we aim to establish a subset of microcephalic dwarfism genes as growth regulators, and thereby further define when and how organism size is determined. These studies will link essential cellular machinery governing proliferation with human disease, identify novel genome-stability factors and may yield insights into the developmental regulation of mammalian size.

Max ERC Funding

2 500 000 €

Duration

Start date: 2018-08-01, End date: 2023-07-31

Project acronymHybReader

ProjectMechanisms of epigenetic gene regulation by R-loops

Researcher (PI)Christof Heinz Niehrs

Host Institution (HI)INSTITUT FUR MOLEKULARE BIOLOGIE GGMBH

Call DetailsAdvanced Grant (AdG), LS2, ERC-2017-ADG

SummaryThe last decade has revolutionized our thinking about regulatory RNAs and yet we are still at the beginning of understanding their biology. One such challenge is presented by R-loops, prevalent DNA:RNA hybrids, which lie at the interface of different nuclear processes, including transcription, RNA processing, lncRNAs, DNA damage, chromatin, and neurodegenerative disease. Long-time considered a threat to genomic integrity, recent evidence indicates that certain R-loops can act as epigenetic gene regulators. The hypothesis is that nascent RNAs retained at their site of transcription may function as sequence-specific component of mammalian chromatin to shape gene expression. Notably, R-loops found at GC-enriched promoters are implicated in preventing DNA methylation and promote transcription, but how this might occur has remained obscure. In a breakthrough discovery, we identified the first epigenetic R-loop reader. This protein binds directly and specifically to DNA:RNA hybrids in vitro and in vivo, and mediates local DNA hydroxymethylation and demethylation via TET (Ten-Eleven Translocation) cytosine oxidases. Using the R-loop reader as a tool provides a unique entry point to address fundamental questions regarding mechanisms and regulation of the epigenetic function and biological role of these DNA:RNA hybrids. Applying genome-wide approaches and studying the biology of embryonic stem cells (ESCs), we will 1) systematically identify regulatory R-loops, 2) characterize their common features, 3) address how R-loops are decoded in ESC pluripotency and differentiation, 4) investigate how regulatory R-loops are erased, and (5) screen for additional R-loop binders/readers and characterize their epigenetic role. The HybReader project will allow for ground-breaking discoveries regarding the emerging epigenetic function and regulation of these poorly understood regulatory hybrids in the control of gene expression.

The last decade has revolutionized our thinking about regulatory RNAs and yet we are still at the beginning of understanding their biology. One such challenge is presented by R-loops, prevalent DNA:RNA hybrids, which lie at the interface of different nuclear processes, including transcription, RNA processing, lncRNAs, DNA damage, chromatin, and neurodegenerative disease. Long-time considered a threat to genomic integrity, recent evidence indicates that certain R-loops can act as epigenetic gene regulators. The hypothesis is that nascent RNAs retained at their site of transcription may function as sequence-specific component of mammalian chromatin to shape gene expression. Notably, R-loops found at GC-enriched promoters are implicated in preventing DNA methylation and promote transcription, but how this might occur has remained obscure. In a breakthrough discovery, we identified the first epigenetic R-loop reader. This protein binds directly and specifically to DNA:RNA hybrids in vitro and in vivo, and mediates local DNA hydroxymethylation and demethylation via TET (Ten-Eleven Translocation) cytosine oxidases. Using the R-loop reader as a tool provides a unique entry point to address fundamental questions regarding mechanisms and regulation of the epigenetic function and biological role of these DNA:RNA hybrids. Applying genome-wide approaches and studying the biology of embryonic stem cells (ESCs), we will 1) systematically identify regulatory R-loops, 2) characterize their common features, 3) address how R-loops are decoded in ESC pluripotency and differentiation, 4) investigate how regulatory R-loops are erased, and (5) screen for additional R-loop binders/readers and characterize their epigenetic role. The HybReader project will allow for ground-breaking discoveries regarding the emerging epigenetic function and regulation of these poorly understood regulatory hybrids in the control of gene expression.

SummaryMutations are the fuel of any evolutionary process, and this also applies to carcinogenesis. The advent of affordable DNA sequencing has enabled mutagenic processes in the human soma to be quantified genome-wide, revealing a striking occurrence of hypermutated tumors. They exhibit an extreme load of somatic changes, often harbouring hundreds of single-nucleotide variants and/or indels per megabase. The HYPER-INSIGHT project is organized into three objectives, which aim to take advantage of the unique opportunity provided by genome sequences of hypermutated and ultramutated tumors. In particular, this work planned in this project aims to further our knowledge on (i) the regional organization of the DNA replication and repair program in human cells, and the determinants thereof, (ii) the extent of selection which acts on somatic variants in various pathways or complexes and (iii) opportunities for selectively targeting DNA repair deficiencies that manifest as hypermutation. Methodologically, our work will employ a three-pronged approach. First, we will perform a multitude of rigorous statistical analyses that draw on the rich and still-expanding resources provided by cancer genomics consortia. Second, we will perform exome and genome sequencing, focusing on ultramutated tumors caused by specific defects in the DNA maintenance machinery. Third, the project involves conditional essentiality screens on cancer cell lines with hypermutant backgrounds. Their goal is to discover synthetic lethality relationships, useful for targeting hypermutating cells, while sparing healthy ones. In summary, one of the promises of cancer genome sequencing projects was to elucidate the mechanisms underlying mutational processes in the human soma, advancing our understanding of this important facet of cancer biology. We will work towards realizing this promise, thereby strengthening the EU’s position in the global scientific endeavour.

Mutations are the fuel of any evolutionary process, and this also applies to carcinogenesis. The advent of affordable DNA sequencing has enabled mutagenic processes in the human soma to be quantified genome-wide, revealing a striking occurrence of hypermutated tumors. They exhibit an extreme load of somatic changes, often harbouring hundreds of single-nucleotide variants and/or indels per megabase. The HYPER-INSIGHT project is organized into three objectives, which aim to take advantage of the unique opportunity provided by genome sequences of hypermutated and ultramutated tumors. In particular, this work planned in this project aims to further our knowledge on (i) the regional organization of the DNA replication and repair program in human cells, and the determinants thereof, (ii) the extent of selection which acts on somatic variants in various pathways or complexes and (iii) opportunities for selectively targeting DNA repair deficiencies that manifest as hypermutation. Methodologically, our work will employ a three-pronged approach. First, we will perform a multitude of rigorous statistical analyses that draw on the rich and still-expanding resources provided by cancer genomics consortia. Second, we will perform exome and genome sequencing, focusing on ultramutated tumors caused by specific defects in the DNA maintenance machinery. Third, the project involves conditional essentiality screens on cancer cell lines with hypermutant backgrounds. Their goal is to discover synthetic lethality relationships, useful for targeting hypermutating cells, while sparing healthy ones. In summary, one of the promises of cancer genome sequencing projects was to elucidate the mechanisms underlying mutational processes in the human soma, advancing our understanding of this important facet of cancer biology. We will work towards realizing this promise, thereby strengthening the EU’s position in the global scientific endeavour.

Max ERC Funding

1 499 813 €

Duration

Start date: 2018-02-01, End date: 2023-01-31

Project acronymIdrSeq

ProjectDiscovery and characterization of functional disordered regions and the genes involved in their regulation through next generation sequencing

Researcher (PI)Madanbabu Mohan

Host Institution (HI)MEDICAL RESEARCH COUNCIL

Call DetailsConsolidator Grant (CoG), LS2, ERC-2015-CoG

SummaryA large fraction of any eukaryotic genome (>40%) encodes protein segments that do not adopt a defined tertiary structure. These proteins or regions are called intrinsically disordered proteins/regions (IDPs/IDRs). IDRs are enriched in critical functions such as transcription and signaling, and have been linked with numerous diseases including neurodegeneration and cancer. In contrast to structured regions, the molecular principles behind the sequence-function relationship of IDRs remain poorly understood.
We propose to identify functional IDRs and discover genes that regulate their function using yeast as a cellular model. We will develop and apply a targeted, high-throughput approach called IdrSeq. This technology exploits next generation sequencing to simultaneously assay vast libraries of sequences (~millions) that code for IDRs by coupling IDR sequence (genotype) to a selectable function (phenotype) and identifying functional variants through a selection experiment.
Specifically, using IdrSeq, we aim to identify and characterize IDRs in a cellular context that can
(Aim 1) activate transcription, and discover genes that regulate IDR mediated transcription
(Aim 2) influence protein stability, and discover genes that regulate IDR mediated half-life
(Aim 3) form higher-order assemblies and discover genes that regulate assembly formation
The unique feature of this proposal is its integrative vision of synthetic & systems biology, structural biology, cell biology, genetics, experiments and computation to establish a discovery platform to study IDRs in a cellular context. Since IdrSeq is modular and scalable, it can be readily extended to investigate a broad range of IDR functions, and adapted to other organisms. Elucidating the principles of sequence-function-gene relationship of IDRs holds enormous potential for synthetic biology. The discovery of genes that regulate IDR function has direct implications for human health by revealing novel therapeutic targets.

A large fraction of any eukaryotic genome (>40%) encodes protein segments that do not adopt a defined tertiary structure. These proteins or regions are called intrinsically disordered proteins/regions (IDPs/IDRs). IDRs are enriched in critical functions such as transcription and signaling, and have been linked with numerous diseases including neurodegeneration and cancer. In contrast to structured regions, the molecular principles behind the sequence-function relationship of IDRs remain poorly understood.
We propose to identify functional IDRs and discover genes that regulate their function using yeast as a cellular model. We will develop and apply a targeted, high-throughput approach called IdrSeq. This technology exploits next generation sequencing to simultaneously assay vast libraries of sequences (~millions) that code for IDRs by coupling IDR sequence (genotype) to a selectable function (phenotype) and identifying functional variants through a selection experiment.
Specifically, using IdrSeq, we aim to identify and characterize IDRs in a cellular context that can
(Aim 1) activate transcription, and discover genes that regulate IDR mediated transcription
(Aim 2) influence protein stability, and discover genes that regulate IDR mediated half-life
(Aim 3) form higher-order assemblies and discover genes that regulate assembly formation
The unique feature of this proposal is its integrative vision of synthetic & systems biology, structural biology, cell biology, genetics, experiments and computation to establish a discovery platform to study IDRs in a cellular context. Since IdrSeq is modular and scalable, it can be readily extended to investigate a broad range of IDR functions, and adapted to other organisms. Elucidating the principles of sequence-function-gene relationship of IDRs holds enormous potential for synthetic biology. The discovery of genes that regulate IDR function has direct implications for human health by revealing novel therapeutic targets.

Max ERC Funding

1 998 126 €

Duration

Start date: 2016-05-01, End date: 2021-04-30

Project acronymLSO

ProjectLiver Spatial Omics

Researcher (PI)Shaul Shalev ITZKOVITZ

Host Institution (HI)WEIZMANN INSTITUTE OF SCIENCE

Call DetailsConsolidator Grant (CoG), LS2, ERC-2017-COG

SummaryThe mammalian liver is a heterogeneous, yet highly structured organ, which performs diverse functions to maintain organismal homeostasis. Hepatocytes operate in repeating hexagonally shaped units termed lobules that are polarized by centripetal blood flow and morphogens. This polarized microenvironment facilitates optimal function by localizing specific processes to distinct lobule layers, a phenomenon known as ‘liver zonation’. While zonation of some key liver functions has been known for years, using spatially resolved single cell transcriptomics, we recently discovered that about 50% of liver genes are zonated. This surprisingly broad spatial heterogeneity raises a fundamental question - do hepatocytes form a uniform population that differs due to spatially graded inputs or are hepatocytes at different zones in fact distinct cell types?
In this proposal we will tackle this question by developing techniques for sorting massive amounts of hepatocytes from defined tissue coordinates at high spatial resolution using zonated surface markers, new zonated reporter mouse models and mRNA content. We will perform a deep and comprehensive profiling of the hepatocyte genome, methylome, epigenome, transcriptome, proteome and metabolome at each zone to characterize liver zonation at all relevant cellular scales. We will also develop an ex-vivo system to functionally characterize the response of hepatocytes from distinct zones to identical input stimuli and the ability of hepatocytes to inter-convert to hepatocytes with differing zonal identities. These experiments will be performed in different metabolic states and along a high fat diet. This project will uncover new features of liver zonation in health and disease and redefine the hepatocyte cell state. Our approach for spatially refined tissue omics can be extended to other structured mammalian organs, thus opening new avenues of research in Systems Biology of mammalian tissues.

The mammalian liver is a heterogeneous, yet highly structured organ, which performs diverse functions to maintain organismal homeostasis. Hepatocytes operate in repeating hexagonally shaped units termed lobules that are polarized by centripetal blood flow and morphogens. This polarized microenvironment facilitates optimal function by localizing specific processes to distinct lobule layers, a phenomenon known as ‘liver zonation’. While zonation of some key liver functions has been known for years, using spatially resolved single cell transcriptomics, we recently discovered that about 50% of liver genes are zonated. This surprisingly broad spatial heterogeneity raises a fundamental question - do hepatocytes form a uniform population that differs due to spatially graded inputs or are hepatocytes at different zones in fact distinct cell types?
In this proposal we will tackle this question by developing techniques for sorting massive amounts of hepatocytes from defined tissue coordinates at high spatial resolution using zonated surface markers, new zonated reporter mouse models and mRNA content. We will perform a deep and comprehensive profiling of the hepatocyte genome, methylome, epigenome, transcriptome, proteome and metabolome at each zone to characterize liver zonation at all relevant cellular scales. We will also develop an ex-vivo system to functionally characterize the response of hepatocytes from distinct zones to identical input stimuli and the ability of hepatocytes to inter-convert to hepatocytes with differing zonal identities. These experiments will be performed in different metabolic states and along a high fat diet. This project will uncover new features of liver zonation in health and disease and redefine the hepatocyte cell state. Our approach for spatially refined tissue omics can be extended to other structured mammalian organs, thus opening new avenues of research in Systems Biology of mammalian tissues.

Max ERC Funding

2 000 000 €

Duration

Start date: 2018-11-01, End date: 2023-10-31

Project acronymLYSOSOMICS

ProjectFunctional Genomics of the Lysosome

Researcher (PI)Andrea BALLABIO

Host Institution (HI)FONDAZIONE TELETHON

Call DetailsAdvanced Grant (AdG), LS2, ERC-2015-AdG

SummaryFor a long time the lysosome has been viewed as a “static” organelle that performs “routine” work for the cell, mostly pertaining to degradation and recycling of cellular waste. My group has challenged this view and used a systems biology approach to discover that the lysosome is subject to a global transcriptional regulation, is able to adapt to environmental clues, and acts as a signalling hub to regulate cell homeostasis. Furthermore, an emerging role of the lysosome has been identified in many types of diseases, including the common neurodegenerative disorders Parkinson's and Alzheimer’s. These findings have opened entirely new fields of investigation on lysosomal biology, suggesting that there is a lot to be learned on the role of the lysosome in health and disease. The goal of LYSOSOMICS is to use “omics” approaches to study lysosomal function and its regulation in normal and pathological conditions. In this “organellar systems biology project” we plan to perform several types of genetic perturbations in three widely used cell lines and study their effects on lysosomal function using a set of newly developed cellular phenotypic assays. Moreover, we plan to identify lysosomal protein-protein interactions using a novel High Content FRET-based approach. Finally, we will use the CRISPR-Cas9 technology to generate a collection of cellular models for all lysosomal storage diseases, a group of severe inherited diseases often associated with early onset neurodegeneration. State-of-the-art computational approaches will be used to predict gene function and identify disease mechanisms potentially exploitable for therapeutic purposes. The physiological relevance of newly identified pathways will be validated by in vivo studies performed on selected genes by using medaka and mice as model systems. This study will allow us to gain a comprehensive understanding of lysosomal function and dysfunction and to use this knowledge to develop new therapeutic strategies.

For a long time the lysosome has been viewed as a “static” organelle that performs “routine” work for the cell, mostly pertaining to degradation and recycling of cellular waste. My group has challenged this view and used a systems biology approach to discover that the lysosome is subject to a global transcriptional regulation, is able to adapt to environmental clues, and acts as a signalling hub to regulate cell homeostasis. Furthermore, an emerging role of the lysosome has been identified in many types of diseases, including the common neurodegenerative disorders Parkinson's and Alzheimer’s. These findings have opened entirely new fields of investigation on lysosomal biology, suggesting that there is a lot to be learned on the role of the lysosome in health and disease. The goal of LYSOSOMICS is to use “omics” approaches to study lysosomal function and its regulation in normal and pathological conditions. In this “organellar systems biology project” we plan to perform several types of genetic perturbations in three widely used cell lines and study their effects on lysosomal function using a set of newly developed cellular phenotypic assays. Moreover, we plan to identify lysosomal protein-protein interactions using a novel High Content FRET-based approach. Finally, we will use the CRISPR-Cas9 technology to generate a collection of cellular models for all lysosomal storage diseases, a group of severe inherited diseases often associated with early onset neurodegeneration. State-of-the-art computational approaches will be used to predict gene function and identify disease mechanisms potentially exploitable for therapeutic purposes. The physiological relevance of newly identified pathways will be validated by in vivo studies performed on selected genes by using medaka and mice as model systems. This study will allow us to gain a comprehensive understanding of lysosomal function and dysfunction and to use this knowledge to develop new therapeutic strategies.

Max ERC Funding

2 362 563 €

Duration

Start date: 2016-10-01, End date: 2021-09-30

Project acronymMEL-Interactions

ProjectAn integrative approach for the exploration of melanoma genetic and immunological interactions

Researcher (PI)Yardena Rachel SAMUELS

Host Institution (HI)WEIZMANN INSTITUTE OF SCIENCE

Call DetailsConsolidator Grant (CoG), LS2, ERC-2017-COG

SummaryTumor development emerges from an accumulation of somatic alterations that together enable malignant growth. These alterations are immensely diverse and the fate of a cell acquiring an alteration may depend on other alterations already present. Despite our progress in mapping the cancer genetic landscape and an expanding catalogue of cancer genes, a need arises to establish how alterations in cancer genes interact to transform healthy cells into cancer cells. Many fundamental questions regarding genomic interactions remain open. For example, we do not know which proteins make up signaling pathway hubs and in which genetic contexts, how genetic alterations interact functionally, how cancer genetic alterations influence the interaction with T cells and how these affect patient response to therapy. The recent growth in the number of genomics data sets gives rise to a parallel increase in statistical power to detect more complex associations allowing robust analyses of complex interrelated genomic networks. In this proposal we suggest to employ our expertise and unique toolsets, to shed new light on the complex interrelated networks formed in melanoma. We propose to combine state-of-the art high content tools with mechanistic studies to discover the structure of signaling-hub organization in melanoma (Aim 1), functionally characterize the complex genetic interactions within the melanoma genome using genome engineering approaches (Aim 2), and to decipher the immuno-genetic interactions between melanoma and T cells (Aim 3). Importantly, we will try to bridge the knowledge gap in deciphering melanoma-specific gene interactions, protein interactions and interactions with T cells by creating new tools and experimental models. Our findings should make an important step towards an unprecedented, thorough and multifaceted understanding of melanoma biology. More broadly, we believe these approaches provide a paradigm for addressing similarly complex questions in other cancers.

Tumor development emerges from an accumulation of somatic alterations that together enable malignant growth. These alterations are immensely diverse and the fate of a cell acquiring an alteration may depend on other alterations already present. Despite our progress in mapping the cancer genetic landscape and an expanding catalogue of cancer genes, a need arises to establish how alterations in cancer genes interact to transform healthy cells into cancer cells. Many fundamental questions regarding genomic interactions remain open. For example, we do not know which proteins make up signaling pathway hubs and in which genetic contexts, how genetic alterations interact functionally, how cancer genetic alterations influence the interaction with T cells and how these affect patient response to therapy. The recent growth in the number of genomics data sets gives rise to a parallel increase in statistical power to detect more complex associations allowing robust analyses of complex interrelated genomic networks. In this proposal we suggest to employ our expertise and unique toolsets, to shed new light on the complex interrelated networks formed in melanoma. We propose to combine state-of-the art high content tools with mechanistic studies to discover the structure of signaling-hub organization in melanoma (Aim 1), functionally characterize the complex genetic interactions within the melanoma genome using genome engineering approaches (Aim 2), and to decipher the immuno-genetic interactions between melanoma and T cells (Aim 3). Importantly, we will try to bridge the knowledge gap in deciphering melanoma-specific gene interactions, protein interactions and interactions with T cells by creating new tools and experimental models. Our findings should make an important step towards an unprecedented, thorough and multifaceted understanding of melanoma biology. More broadly, we believe these approaches provide a paradigm for addressing similarly complex questions in other cancers.

Max ERC Funding

2 000 000 €

Duration

Start date: 2018-10-01, End date: 2023-09-30

Project acronymMETACELL

ProjectMetabolism of a cell pictured by single-cell approach

Researcher (PI)Theodore Alexandrov

Host Institution (HI)EUROPEAN MOLECULAR BIOLOGY LABORATORY

Call DetailsConsolidator Grant (CoG), LS2, ERC-2017-COG

SummaryEvery cell is unique. Metabolites define the composition of each cell and play key roles in essential intracellular processes of energy production and uptake, signaling, regulation, and cell death. Obtaining metabolite signatures of individual cells and linking them to cellular phenotypes is of paramount importance for a holistic understanding of these processes. This requires high-throughput single-cell metabolomics that is not generally attainable due to the limited sensitivity, low throughput, and disruptiveness of state-of-the-art metabolomics methods.
I propose to develop a spatial single-cell metabolomics approach for human cell culture systems. The approach will be based on using metabolite imaging mass spectrometry and will provide metabolite profiles of individual cells and metabolite signatures of single-cell phenotypes identified by light microscopy. With this approach developed, I will investigate the link between the intracellular metabolism and single-cell phenotype and focus on the following questions: How is the intracellular metabolism linked to cellular heterogeneity? How high is the variation of essential metabolites in a cell population? How do the energy metabolism and lipids biosynthesis change through the cell cycle and infection stages? What is the metabolic response to inflammatory signals?
I will scale up the analysis to discover novel cell phenotypes both in the cell culture systems and in big metabolite imaging mass spectrometry data from various biological systems provided to us by our collaborators and the community, and representing billions of cells.
My project will enable spatial single-cell metabolomics on a large scale and will provide yet lacking capacity for investigating and visualizing the intracellular metabolism on a single-cell level. It will advance our molecular understanding of key biological processes and pave the way to discoveries of molecular mechanisms of inflammation, cancer, and infection.

Every cell is unique. Metabolites define the composition of each cell and play key roles in essential intracellular processes of energy production and uptake, signaling, regulation, and cell death. Obtaining metabolite signatures of individual cells and linking them to cellular phenotypes is of paramount importance for a holistic understanding of these processes. This requires high-throughput single-cell metabolomics that is not generally attainable due to the limited sensitivity, low throughput, and disruptiveness of state-of-the-art metabolomics methods.
I propose to develop a spatial single-cell metabolomics approach for human cell culture systems. The approach will be based on using metabolite imaging mass spectrometry and will provide metabolite profiles of individual cells and metabolite signatures of single-cell phenotypes identified by light microscopy. With this approach developed, I will investigate the link between the intracellular metabolism and single-cell phenotype and focus on the following questions: How is the intracellular metabolism linked to cellular heterogeneity? How high is the variation of essential metabolites in a cell population? How do the energy metabolism and lipids biosynthesis change through the cell cycle and infection stages? What is the metabolic response to inflammatory signals?
I will scale up the analysis to discover novel cell phenotypes both in the cell culture systems and in big metabolite imaging mass spectrometry data from various biological systems provided to us by our collaborators and the community, and representing billions of cells.
My project will enable spatial single-cell metabolomics on a large scale and will provide yet lacking capacity for investigating and visualizing the intracellular metabolism on a single-cell level. It will advance our molecular understanding of key biological processes and pave the way to discoveries of molecular mechanisms of inflammation, cancer, and infection.

Max ERC Funding

2 330 628 €

Duration

Start date: 2018-07-01, End date: 2023-06-30

Project acronymmiRCell

ProjectMicroRNA functions in single cells

Researcher (PI)Marc FRIEDLÄNDER

Host Institution (HI)STOCKHOLMS UNIVERSITET

Call DetailsStarting Grant (StG), LS2, ERC-2017-STG

SummaryIt is now becoming apparent that genes are regulated not only by transcription, but also by thousands of post-transcriptional regulators that can stabilize or degrade mRNAs. Some of the most important regulators are miRNAs, short RNA molecules that are deeply conserved in sequence and are involved in numerous biological processes, including human disease. Surprisingly, transcriptomic and proteomic studies show that most miRNAs only have subtle silencing effects on their targets, suggesting additional important, but yet undiscovered functions. Thus the question is raised: if the main function of miRNAs is not to silence targets, what is it?
I will test two novel hypotheses about miRNA function. The first hypothesis proposes that miRNAs can buffer gene expression noise. The second hypothesis is inspired by my preliminary results and proposes that miRNAs can synchronize expression of genes. If I validate either hypothesis, it would mean that miRNA functions can be investigated in entirely new ways, yielding important new biological insights relevant to both basic research and human health. However, these hypotheses can only be tested in individual cells, and the necessary single-cell technologies and computational tools are only maturing now.
I will apply my expertise in miRNA biology and in combined wet-lab and computational methods to design, develop and apply miRCell-seq to test these two hypotheses in cell cultures and in animals. This new method will for the first time measure miRNAs, their targets, and the interactions between them in single cells and transcriptome-wide. We will use mutant cells devoid of miRNAs and time course experiments to generate sufficient data to develop detailed models of the miRNA impact on their targets. We will then validate our findings with single cell proteomics. This project thus has the potential to reveal novel functions of miRNAs and substantially improve our general understanding of gene regulation.

It is now becoming apparent that genes are regulated not only by transcription, but also by thousands of post-transcriptional regulators that can stabilize or degrade mRNAs. Some of the most important regulators are miRNAs, short RNA molecules that are deeply conserved in sequence and are involved in numerous biological processes, including human disease. Surprisingly, transcriptomic and proteomic studies show that most miRNAs only have subtle silencing effects on their targets, suggesting additional important, but yet undiscovered functions. Thus the question is raised: if the main function of miRNAs is not to silence targets, what is it?
I will test two novel hypotheses about miRNA function. The first hypothesis proposes that miRNAs can buffer gene expression noise. The second hypothesis is inspired by my preliminary results and proposes that miRNAs can synchronize expression of genes. If I validate either hypothesis, it would mean that miRNA functions can be investigated in entirely new ways, yielding important new biological insights relevant to both basic research and human health. However, these hypotheses can only be tested in individual cells, and the necessary single-cell technologies and computational tools are only maturing now.
I will apply my expertise in miRNA biology and in combined wet-lab and computational methods to design, develop and apply miRCell-seq to test these two hypotheses in cell cultures and in animals. This new method will for the first time measure miRNAs, their targets, and the interactions between them in single cells and transcriptome-wide. We will use mutant cells devoid of miRNAs and time course experiments to generate sufficient data to develop detailed models of the miRNA impact on their targets. We will then validate our findings with single cell proteomics. This project thus has the potential to reveal novel functions of miRNAs and substantially improve our general understanding of gene regulation.

SummaryAdvances in DNA sequencing technology, enabling routine genetic variation studies, have uncovered that genomic structural variants (SVs; e.g. deletions and inversions) account for most varying bases in human genomes. SVs are also disproportionally associated with disease phenotypes when compared to single nucleotide variants by number. Studies are increasingly implicating genetic polymorphisms with diseases – yet why some humans develop diseases while others do not, and why disease incidences often increase with age, is largely unclear.
Intriguingly, recent studies showed that human genetic variation extends markedly beyond heritable variants. Soon after fertilization, mutations naturally accumulate in healthy tissues resulting in somatic genetic mosaicism (SGM), a highly understudied form of variation. Among SGM classes, ‘SV mosaicisms’ likely account for most varying bases, are increased at age, are seen in the context of clonal cell expansion, and are associated with diseases of the elderly including type 2 diabetes and cancer. This indicates that to understand the basis of particular diseases we may first need to comprehend how naturally formed somatic SVs impact human cells.
Here we aim to uncover the extent and impact of SV mosaicism. We aim to pursue single cell analyses, which offer the most direct way to detect somatic SVs in individual cells. Performing SV analysis in single cells at scale, however, is not a mainstream approach: current methods identify copy-number variants (CNVs), but miss key copy-neutral SV classes (e.g. inversions) likely to be highly relevant. We aim to develop new experimental and computational tools to construct a single cell catalog of a wide variety of relevant SV classes in different cell types (i.e. the blood compartment and skin) and ages. Using this catalog, we aim to study the functional impact of SV mosaicism on the cellular level, as a foundation for elucidating roles of somatic SVs in age-related phenotypes and diseases.

Advances in DNA sequencing technology, enabling routine genetic variation studies, have uncovered that genomic structural variants (SVs; e.g. deletions and inversions) account for most varying bases in human genomes. SVs are also disproportionally associated with disease phenotypes when compared to single nucleotide variants by number. Studies are increasingly implicating genetic polymorphisms with diseases – yet why some humans develop diseases while others do not, and why disease incidences often increase with age, is largely unclear.
Intriguingly, recent studies showed that human genetic variation extends markedly beyond heritable variants. Soon after fertilization, mutations naturally accumulate in healthy tissues resulting in somatic genetic mosaicism (SGM), a highly understudied form of variation. Among SGM classes, ‘SV mosaicisms’ likely account for most varying bases, are increased at age, are seen in the context of clonal cell expansion, and are associated with diseases of the elderly including type 2 diabetes and cancer. This indicates that to understand the basis of particular diseases we may first need to comprehend how naturally formed somatic SVs impact human cells.
Here we aim to uncover the extent and impact of SV mosaicism. We aim to pursue single cell analyses, which offer the most direct way to detect somatic SVs in individual cells. Performing SV analysis in single cells at scale, however, is not a mainstream approach: current methods identify copy-number variants (CNVs), but miss key copy-neutral SV classes (e.g. inversions) likely to be highly relevant. We aim to develop new experimental and computational tools to construct a single cell catalog of a wide variety of relevant SV classes in different cell types (i.e. the blood compartment and skin) and ages. Using this catalog, we aim to study the functional impact of SV mosaicism on the cellular level, as a foundation for elucidating roles of somatic SVs in age-related phenotypes and diseases.

Max ERC Funding

1 997 060 €

Duration

Start date: 2019-02-01, End date: 2024-01-31

Project acronymMultiCellSysBio

ProjectDeconstructing complexity to reveal quantitative systems-level principles that enable multicellular systems to coordinately regulate genes over space and time

Researcher (PI)Hyun Oh Youk

Host Institution (HI)TECHNISCHE UNIVERSITEIT DELFT

Call DetailsStarting Grant (StG), LS2, ERC-2015-STG

SummaryA key question in biology is how cells at different locations communicate through signalling molecules so that cells at the right place and time turn on the right genes. Such coordination is vital for many processes including the development of all embryos. A common set of strategies that cells from distinct organisms use to coordinate their gene expression has long been elusive. Finding it requires quantifying how two key factors, the spatial locations of cells and the genetic circuits that control the cells’ secretion of signalling molecules, each affects cells' gene expressions. This has been challenging because their effects are often intertwined in complex ways.
To overcome this challenge, I will assemble budding yeasts into multicellular structures component-by-component in a bottom-up manner, from building genetic circuits to arranging cells in space. I will build genetic circuits whose motifs commonly occur in natural systems. The yeasts will use the genetic circuits to control secretion and sensing of three distinct signalling molecules for communication. Using adhesive proteins and light-inducible genes, I will assemble multiple yeast strains, each with a unique genetic circuit, into a single two- and three-dimensional multicellular structure. The structures will mimic various sizes, shapes, and arrangements of cells found in nature. I will then switch on the circuits in these cells to initiate communication between cells. Different amounts of signalling molecules will cause the cells to make different amounts of fluorescent proteins. By measuring the fluorescence of cells at different locations over time and then correlating them, I will infer the degree of cell-cell coordination. I will build mathematical models to guide the experiments. By finding which combinations of genetic circuits and spatial arrangements of cells enable cell-cell coordination of gene expressions, I will reveal design principles of multicellular systems that have been elusive.

A key question in biology is how cells at different locations communicate through signalling molecules so that cells at the right place and time turn on the right genes. Such coordination is vital for many processes including the development of all embryos. A common set of strategies that cells from distinct organisms use to coordinate their gene expression has long been elusive. Finding it requires quantifying how two key factors, the spatial locations of cells and the genetic circuits that control the cells’ secretion of signalling molecules, each affects cells' gene expressions. This has been challenging because their effects are often intertwined in complex ways.
To overcome this challenge, I will assemble budding yeasts into multicellular structures component-by-component in a bottom-up manner, from building genetic circuits to arranging cells in space. I will build genetic circuits whose motifs commonly occur in natural systems. The yeasts will use the genetic circuits to control secretion and sensing of three distinct signalling molecules for communication. Using adhesive proteins and light-inducible genes, I will assemble multiple yeast strains, each with a unique genetic circuit, into a single two- and three-dimensional multicellular structure. The structures will mimic various sizes, shapes, and arrangements of cells found in nature. I will then switch on the circuits in these cells to initiate communication between cells. Different amounts of signalling molecules will cause the cells to make different amounts of fluorescent proteins. By measuring the fluorescence of cells at different locations over time and then correlating them, I will infer the degree of cell-cell coordination. I will build mathematical models to guide the experiments. By finding which combinations of genetic circuits and spatial arrangements of cells enable cell-cell coordination of gene expressions, I will reveal design principles of multicellular systems that have been elusive.

Max ERC Funding

1 500 000 €

Duration

Start date: 2016-04-01, End date: 2021-03-31

Project acronymNonChroRep

ProjectInvestigating the role of the long noncoding transcriptome in chromatin replication

SummaryA major shift in our conception of genome regulation has emerged in recent years. It is now obvious that the majority of cellular transcripts do not code for proteins, and a significant subset of them are long RNAs (lncRNAs). My lab and others have shown that lncRNAs regulate genome function and gene expression, and that alterations in lncRNAs are inherent to disease, including cancer. However, our understanding of the roles of lncRNAs and their underlying molecular mechanisms are still extremely poor.
Among all the mechanisms reported, the evident connection between lncRNAs and the chromatin places them at the center of cell biology. During their cycle, cells must undergo faithful DNA replication to ensure that an exact copy of their genetic content is passed on to their daughters. Throughout this tightly regulated process chromatin must be disrupted and reconstituted, and it determines where and when replication takes place. If replication is deregulated, cells can proliferate uncontrollably and suffer loss of genome integrity. Our recent findings implicate lncRNA in the process of DNA replication, representing a novel aspect of genome regulation that places lncRNAs at the focal point of cancer biology. To delve deeper into these findings I aim to:
1. Identify the role of lncRNAs in the replication of the chromatin
2. Dissect the molecular mechanism by which lncRNAs function in this process and
3. Explore the role of these lncRNAs as cancer drivers and their potential as therapeutic targets.
I will apply tools that we have generated in recent years, as well as new ones, including approaches to identify lncRNAs associated with replicating chromatin, novel lncRNA-tailored CRISPR applications, and the latest methodology for functional study and targeting of long noncoding transcripts in cancer. I am confident that we are in a unique position to address these life-essential and yet pending questions, setting up a basis for future lncRNA-based therapies.

A major shift in our conception of genome regulation has emerged in recent years. It is now obvious that the majority of cellular transcripts do not code for proteins, and a significant subset of them are long RNAs (lncRNAs). My lab and others have shown that lncRNAs regulate genome function and gene expression, and that alterations in lncRNAs are inherent to disease, including cancer. However, our understanding of the roles of lncRNAs and their underlying molecular mechanisms are still extremely poor.
Among all the mechanisms reported, the evident connection between lncRNAs and the chromatin places them at the center of cell biology. During their cycle, cells must undergo faithful DNA replication to ensure that an exact copy of their genetic content is passed on to their daughters. Throughout this tightly regulated process chromatin must be disrupted and reconstituted, and it determines where and when replication takes place. If replication is deregulated, cells can proliferate uncontrollably and suffer loss of genome integrity. Our recent findings implicate lncRNA in the process of DNA replication, representing a novel aspect of genome regulation that places lncRNAs at the focal point of cancer biology. To delve deeper into these findings I aim to:
1. Identify the role of lncRNAs in the replication of the chromatin
2. Dissect the molecular mechanism by which lncRNAs function in this process and
3. Explore the role of these lncRNAs as cancer drivers and their potential as therapeutic targets.
I will apply tools that we have generated in recent years, as well as new ones, including approaches to identify lncRNAs associated with replicating chromatin, novel lncRNA-tailored CRISPR applications, and the latest methodology for functional study and targeting of long noncoding transcripts in cancer. I am confident that we are in a unique position to address these life-essential and yet pending questions, setting up a basis for future lncRNA-based therapies.

SummaryFinding the mutations, genes and pathways directly involved in cancer is of paramount importance to understand the mechanisms of tumour development and devise therapeutic strategies to overcome the disease. Due to their role in cancer development and maintenance, the proteins encoded by cancer genes are candidate therapeutic targets. Indeed, in recent years we have witnessed the development of successful cancer-targeting therapies to counteract the effect of driver mutations. Although the coding part of the human genome has now largely been explored in the search for cancer driver mutations in most frequent cancer types, the extent of involvement of noncoding mutations in cancer development remains a mystery. The main challenges faced are: 1) the functional role of most noncoding regions is unknown, and 2) tumours often have thousands of somatic mutations, so that distinguishing cancer driver mutations from bystanders is like finding the proverbial needle in a haystack. To overcome these two challenges I propose to analyse the pattern of somatic mutations across thousands of tumours in noncoding regions to identify signals of positive selection. These signals are an indication that mutations in the region have been positively selected during tumour evolution and are thus directly involved in the tumour phenotype. The large scale analysis proposed here will allow us to create a catalogue of noncoding elements involved in different types of cancer upon mutations. We will study in detail a selected set of driver elements to uncover their specific function and role in the tumourigenic process. Furthermore, we will explore possibilities of counteracting their driver effect with targeted drugs. The results of this project may boost our understanding of the biological role of noncoding regions, help to unravel novel molecular causes of cancer and provide novel targeted therapeutic opportunities for cancer patients.

Finding the mutations, genes and pathways directly involved in cancer is of paramount importance to understand the mechanisms of tumour development and devise therapeutic strategies to overcome the disease. Due to their role in cancer development and maintenance, the proteins encoded by cancer genes are candidate therapeutic targets. Indeed, in recent years we have witnessed the development of successful cancer-targeting therapies to counteract the effect of driver mutations. Although the coding part of the human genome has now largely been explored in the search for cancer driver mutations in most frequent cancer types, the extent of involvement of noncoding mutations in cancer development remains a mystery. The main challenges faced are: 1) the functional role of most noncoding regions is unknown, and 2) tumours often have thousands of somatic mutations, so that distinguishing cancer driver mutations from bystanders is like finding the proverbial needle in a haystack. To overcome these two challenges I propose to analyse the pattern of somatic mutations across thousands of tumours in noncoding regions to identify signals of positive selection. These signals are an indication that mutations in the region have been positively selected during tumour evolution and are thus directly involved in the tumour phenotype. The large scale analysis proposed here will allow us to create a catalogue of noncoding elements involved in different types of cancer upon mutations. We will study in detail a selected set of driver elements to uncover their specific function and role in the tumourigenic process. Furthermore, we will explore possibilities of counteracting their driver effect with targeted drugs. The results of this project may boost our understanding of the biological role of noncoding regions, help to unravel novel molecular causes of cancer and provide novel targeted therapeutic opportunities for cancer patients.

Max ERC Funding

1 995 829 €

Duration

Start date: 2016-12-01, End date: 2021-11-30

Project acronymNucleolusChromatin

ProjectAnalysis of the nucleolus in genome organization and function

Researcher (PI)Raffaella SANTORO

Host Institution (HI)UNIVERSITAT ZURICH

Call DetailsAdvanced Grant (AdG), LS2, ERC-2017-ADG

SummaryIn eukaryotic cells, the higher-order organization of genomes is functionally important to ensure correct execution of gene expression programs. For instance, as cells differentiate into specialized cell types, chromosomes undergo diverse structural and organizational changes that affect gene expression and other cellular functions. However, how this process is achieved is still poorly understood. The elucidation of the mechanisms that control the spatial architecture of the genome and its contribution to gene regulation is a key open issue in molecular biology, relevant for physiological and pathological processes.
Increasing evidence indicated that large-scale folding of chromatin may affect gene expression by locating genes to specific nuclear subcompartments that are either stimulatory or inhibitory to transcription. Nuclear periphery (NP) and nucleolus are two important nuclear landmarks where repressive chromatin domains are often located. The interaction of chromosomes with NP and nucleolus is thought to contribute to a basal chromosome architecture and genome function. However, while the role of NP in genome organization has been well documented, the function of the nucleolus remains yet elusive.
To fully understand how genome organization regulates chromatin and gene expression states, it is necessary to obtain a comprehensive functional map of genome compartmentalization. However, so far, only domains associating with NP (LADs) have been identified and characterized while nucleolar-associated domains (NADs) remained under-investigated. The aim of this project is to fill this gap by developing methods to identify and characterize NADs and analyse the role of the nucleolus in genome organization, moving toward the obtainment of a comprehensive functional map of genome compartmentalization for each cell state and providing novel insights into basic principles of genome organization and its role in gene expression and cell function that yet remain elusive.

In eukaryotic cells, the higher-order organization of genomes is functionally important to ensure correct execution of gene expression programs. For instance, as cells differentiate into specialized cell types, chromosomes undergo diverse structural and organizational changes that affect gene expression and other cellular functions. However, how this process is achieved is still poorly understood. The elucidation of the mechanisms that control the spatial architecture of the genome and its contribution to gene regulation is a key open issue in molecular biology, relevant for physiological and pathological processes.
Increasing evidence indicated that large-scale folding of chromatin may affect gene expression by locating genes to specific nuclear subcompartments that are either stimulatory or inhibitory to transcription. Nuclear periphery (NP) and nucleolus are two important nuclear landmarks where repressive chromatin domains are often located. The interaction of chromosomes with NP and nucleolus is thought to contribute to a basal chromosome architecture and genome function. However, while the role of NP in genome organization has been well documented, the function of the nucleolus remains yet elusive.
To fully understand how genome organization regulates chromatin and gene expression states, it is necessary to obtain a comprehensive functional map of genome compartmentalization. However, so far, only domains associating with NP (LADs) have been identified and characterized while nucleolar-associated domains (NADs) remained under-investigated. The aim of this project is to fill this gap by developing methods to identify and characterize NADs and analyse the role of the nucleolus in genome organization, moving toward the obtainment of a comprehensive functional map of genome compartmentalization for each cell state and providing novel insights into basic principles of genome organization and its role in gene expression and cell function that yet remain elusive.

Max ERC Funding

2 500 000 €

Duration

Start date: 2018-09-01, End date: 2023-08-31

Project acronymORGANOMICS

ProjectReconstructing human cortex development and malformation with single-cell transcriptomics

SummaryTechnologies to sequence single-cell transcriptomes (scRNA-seq) are revolutionizing our ability to analyze cell composition and differentiation in complex tissues. In parallel, recent innovations allow the generation of three-dimensional tissues from stem cells that recapitulate human development. In this proposal, we will focus on human cortex development modelled by cerebral organoids. Our vision is to create an integrative single-cell transcriptomic platform to reconstruct cerebral organoid development, and dissect network alterations that lead to human brain malformations. Our project will be advanced through the following objectives:
1. Single-cell transcriptome-coupled lineage tracing: We will use cellular barcoding to label individual cortical progenitor cells, trace their output and lineage trees with high-throughput scRNA-seq, and quantify lineage transition probabilities between cell types.
2. Gene knockout screens in mosaic organoids: We will use CRISPR/Cas9 to perform genetic screens of up to 100 genotypes in mosaic organoids to understand mechanisms that regulate cell lineage decisions during cortex development.
3. High-throughput reconstructions of cortex malformations: We will generate cerebral organoids from patients with cortical malformations and reconstruct networks and infer differentiation hierarchies using high-throughput and lineage-coupled scRNA-seq. We will spatially resolve network aberrations using sequential fluorescence in situ hybridization (seqFISH).
ORGANOMICS provides an entirely new quantitative direction to study human corticogenesis. We will build high-resolution models of cortex development by measuring the expression and function of genes in thousands of single cells. Our interdisciplinary project will lead to groundbreaking insight into mechanisms underlying neurodevelopmental diseases. Our general strategy can be extended to various other organ systems where protocols to generate in vitro counterparts can be established.

Technologies to sequence single-cell transcriptomes (scRNA-seq) are revolutionizing our ability to analyze cell composition and differentiation in complex tissues. In parallel, recent innovations allow the generation of three-dimensional tissues from stem cells that recapitulate human development. In this proposal, we will focus on human cortex development modelled by cerebral organoids. Our vision is to create an integrative single-cell transcriptomic platform to reconstruct cerebral organoid development, and dissect network alterations that lead to human brain malformations. Our project will be advanced through the following objectives:
1. Single-cell transcriptome-coupled lineage tracing: We will use cellular barcoding to label individual cortical progenitor cells, trace their output and lineage trees with high-throughput scRNA-seq, and quantify lineage transition probabilities between cell types.
2. Gene knockout screens in mosaic organoids: We will use CRISPR/Cas9 to perform genetic screens of up to 100 genotypes in mosaic organoids to understand mechanisms that regulate cell lineage decisions during cortex development.
3. High-throughput reconstructions of cortex malformations: We will generate cerebral organoids from patients with cortical malformations and reconstruct networks and infer differentiation hierarchies using high-throughput and lineage-coupled scRNA-seq. We will spatially resolve network aberrations using sequential fluorescence in situ hybridization (seqFISH).
ORGANOMICS provides an entirely new quantitative direction to study human corticogenesis. We will build high-resolution models of cortex development by measuring the expression and function of genes in thousands of single cells. Our interdisciplinary project will lead to groundbreaking insight into mechanisms underlying neurodevelopmental diseases. Our general strategy can be extended to various other organ systems where protocols to generate in vitro counterparts can be established.

SummaryThe perpetual arms race between bacteria and phage has resulted in the evolution of efficient resistance systems that protect bacteria from phage infection. Such systems, which include restriction enzymes and CRISPR-Cas, have major influence on the evolution of both bacteria and phage, and have also proven to be invaluable for molecular and biotechnological applications. Although much have been learned on the biology of bacterial defense against phage, more than half of all sequenced bacteria do not contain CRISPR-Cas, and it is estimated that many additional, yet-uncharacterized anti-phage defense systems are encoded in bacterial genomes.
The goal of this project is to systematically understand the arsenal of defense mechanisms that are at the disposal of microbes in their struggle against phages. The project combines computational genomics, synthetic biology, high-throughput robotic assays, and deep genetic and biochemical experiments to discover, verify, and study the properties of anti-phage defense systems.

The perpetual arms race between bacteria and phage has resulted in the evolution of efficient resistance systems that protect bacteria from phage infection. Such systems, which include restriction enzymes and CRISPR-Cas, have major influence on the evolution of both bacteria and phage, and have also proven to be invaluable for molecular and biotechnological applications. Although much have been learned on the biology of bacterial defense against phage, more than half of all sequenced bacteria do not contain CRISPR-Cas, and it is estimated that many additional, yet-uncharacterized anti-phage defense systems are encoded in bacterial genomes.
The goal of this project is to systematically understand the arsenal of defense mechanisms that are at the disposal of microbes in their struggle against phages. The project combines computational genomics, synthetic biology, high-throughput robotic assays, and deep genetic and biochemical experiments to discover, verify, and study the properties of anti-phage defense systems.

Max ERC Funding

2 000 000 €

Duration

Start date: 2016-07-01, End date: 2021-06-30

Project acronymPIWI-Chrom

ProjectUnderstanding small RNA-mediated transposon control at the level of chromatin in the animal germline

Researcher (PI)Julius Brennecke

Host Institution (HI)INSTITUT FUER MOLEKULARE BIOTECHNOLOGIE GMBH

Call DetailsConsolidator Grant (CoG), LS2, ERC-2015-CoG

SummaryTransposable elements—universal components of genomes—pose a major threat to genome integrity due to their mutagenic character. In all eukaryotic lineages small RNA pathways act as defense systems to protect the host genome against the activity of transposons. The central pathway in animals is the gonad-specific PIWI interacting RNA (piRNA) pathway, one of the most elaborate but also least understood small RNA silencing systems.
Here I propose to study the interplay between the piRNA pathway and chromatin biology in Drosophila with two aims: First, we will identify the factors and investigate the processes that underlie piRNA-guided silencing in the nucleus. Our objective is to understand how recruitment of an Argonaute protein to a nascent RNA mechanistically leads to the assembly of effector proteins that govern heterochromatin formation and transcriptional silencing. Second, we will study the biology of piRNA clusters, heterochromatic loci that encompass a library of transposon fragments and that act as the pathway’s memory system. Our goal is to uncover how a group of proteins—several of which are germline-specific variants of basic cellular factors—enable transcription within heterochromatin and control the downstream fate of the emerging non-coding RNAs.
Our work centers on the piRNA pathway in Drosophila ovaries, undeniably the model system at the forefront of the field. By combining the strength of fly genetics with the power of genome-wide approaches we will uncover how heterochromatin on the one hand governs silencing and how the piRNA pathway on the other hand exploits it to facilitate the transcription of piRNA precursors. This will reveal fundamental insights into the co-evolution of transposons and host genomes. At the same time, by studying the piRNA pathway’s intersection with chromatin biology and transcription, we expect to reveal new insights into basic principles of gene expression.

Transposable elements—universal components of genomes—pose a major threat to genome integrity due to their mutagenic character. In all eukaryotic lineages small RNA pathways act as defense systems to protect the host genome against the activity of transposons. The central pathway in animals is the gonad-specific PIWI interacting RNA (piRNA) pathway, one of the most elaborate but also least understood small RNA silencing systems.
Here I propose to study the interplay between the piRNA pathway and chromatin biology in Drosophila with two aims: First, we will identify the factors and investigate the processes that underlie piRNA-guided silencing in the nucleus. Our objective is to understand how recruitment of an Argonaute protein to a nascent RNA mechanistically leads to the assembly of effector proteins that govern heterochromatin formation and transcriptional silencing. Second, we will study the biology of piRNA clusters, heterochromatic loci that encompass a library of transposon fragments and that act as the pathway’s memory system. Our goal is to uncover how a group of proteins—several of which are germline-specific variants of basic cellular factors—enable transcription within heterochromatin and control the downstream fate of the emerging non-coding RNAs.
Our work centers on the piRNA pathway in Drosophila ovaries, undeniably the model system at the forefront of the field. By combining the strength of fly genetics with the power of genome-wide approaches we will uncover how heterochromatin on the one hand governs silencing and how the piRNA pathway on the other hand exploits it to facilitate the transcription of piRNA precursors. This will reveal fundamental insights into the co-evolution of transposons and host genomes. At the same time, by studying the piRNA pathway’s intersection with chromatin biology and transcription, we expect to reveal new insights into basic principles of gene expression.

Max ERC Funding

1 999 530 €

Duration

Start date: 2016-07-01, End date: 2021-06-30

Project acronymPolyDomFormFuncReg

ProjectDiscovering how polycomb domains form and function in gene regulation

Researcher (PI)Robert John Klose

Host Institution (HI)THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD

Call DetailsConsolidator Grant (CoG), LS2, ERC-2015-CoG

SummaryPolycomb group chromatin modifying systems are essential for normal gene regulation and development, and alterations in their activity are a hallmark of a broad range of cancers. Although the chromatin modifications placed by the two central polycomb protein complexes (PRC1 and PRC2) are well-characterized, how this fascinating system selects its target sites in vivo, and then forms polycomb chromatin domains that are repressive to transcription remains enigmatic. This constitutes the major conceptual gap in our understanding of this essential gene regulatory system. We recently discovered a new pathway that is sufficient in model systems to initiate polycomb chromatin domain formation. Building on this discovery, an ambitious high-risk/high-reward yet hypothesis-driven multidisciplinary approach integrating biochemical, molecular, genomic, and single-cell analyses will be exploited to discover the fundamental principles that underpin polycomb domain formation and subsequently transcriptional repression. Specifically, the three aims of the research programme are to: (i) Discover how the KDM2B/PRC1 complex initiates polycomb domain formation, (ii) Discover how polycomb target sites are selected and polycomb domains formed during normal cell lineage commitment, and (iii) Discover how polycomb domains regulate gene expression. Going well beyond the state-of-the-art, our innovative approaches will lead to major new breakthroughs closing the conceptual gap that currently limits our understanding of how polycomb complexes regulate gene expression, an essential first step towards the possibility of devising strategies for therapeutic intervention in human cancers and other diseases where these systems are perturbed. Furthermore, support from the ERC in tackling these important problems will allow me to recruit the talented individuals necessary to achieve our objectives and consolidate my position as an emerging leader in the field.

Polycomb group chromatin modifying systems are essential for normal gene regulation and development, and alterations in their activity are a hallmark of a broad range of cancers. Although the chromatin modifications placed by the two central polycomb protein complexes (PRC1 and PRC2) are well-characterized, how this fascinating system selects its target sites in vivo, and then forms polycomb chromatin domains that are repressive to transcription remains enigmatic. This constitutes the major conceptual gap in our understanding of this essential gene regulatory system. We recently discovered a new pathway that is sufficient in model systems to initiate polycomb chromatin domain formation. Building on this discovery, an ambitious high-risk/high-reward yet hypothesis-driven multidisciplinary approach integrating biochemical, molecular, genomic, and single-cell analyses will be exploited to discover the fundamental principles that underpin polycomb domain formation and subsequently transcriptional repression. Specifically, the three aims of the research programme are to: (i) Discover how the KDM2B/PRC1 complex initiates polycomb domain formation, (ii) Discover how polycomb target sites are selected and polycomb domains formed during normal cell lineage commitment, and (iii) Discover how polycomb domains regulate gene expression. Going well beyond the state-of-the-art, our innovative approaches will lead to major new breakthroughs closing the conceptual gap that currently limits our understanding of how polycomb complexes regulate gene expression, an essential first step towards the possibility of devising strategies for therapeutic intervention in human cancers and other diseases where these systems are perturbed. Furthermore, support from the ERC in tackling these important problems will allow me to recruit the talented individuals necessary to achieve our objectives and consolidate my position as an emerging leader in the field.

Max ERC Funding

1 999 416 €

Duration

Start date: 2016-06-01, End date: 2021-05-31

Project acronymProCovar

ProjectExploring new applications of amino acid covariation analysis in modelling proteins and their complexes

Researcher (PI)David JONES

Host Institution (HI)UNIVERSITY COLLEGE LONDON

Call DetailsAdvanced Grant (AdG), LS2, ERC-2015-AdG

SummaryAs a result of the rapid development of next generation sequencing, we now have access to hundreds and often many thousands of sequences which belong to the same family. Such a large amount of sequence data for a particular protein family, along with recent developments in computational statistics, enables an entirely new kind of evolutionary analysis to be performed on sequences, where for the first time we can compute statistically significant networks of correlated mutations. The proposal describes an integrated programme of work to fully explore the potential applications of the new amino acid covariation techniques in predicting aspects of protein structure and function. A particular emphasis in this proposal are proteins which are difficult to study by experimental techniques i.e. disordered proteins, transmembrane proteins and large complexes. The first objective will be to explore key developments in the underpinning algorithms, tackling both the issue of needing very large numbers of homologous sequences and also the downstream 3-D embedding to produce viable models. The second objective will involve experimental work with a collaborator where the idea that de novo protein design techniques might be exploited to artificially expand the set of available sequences for a given proto-family will be explored. The third objective will focus specifically on transmembrane protein modelling, where covariation-based approaches have proven to be highly effective. Here the goal will be to extend our existing FILM3 method to encompass both beta-barrel type TM proteins, but also to try to handle the issue of homomultimers, which is a critical aspect of TM protein modelling as so many families are known to adopt higher orders of structure than the fold level alone. Finally, applications of covariation analysis to probing multiple conformations of disordered proteins will be developed, with a specific focus on interactions of disordered proteins with DNA and RNA.

As a result of the rapid development of next generation sequencing, we now have access to hundreds and often many thousands of sequences which belong to the same family. Such a large amount of sequence data for a particular protein family, along with recent developments in computational statistics, enables an entirely new kind of evolutionary analysis to be performed on sequences, where for the first time we can compute statistically significant networks of correlated mutations. The proposal describes an integrated programme of work to fully explore the potential applications of the new amino acid covariation techniques in predicting aspects of protein structure and function. A particular emphasis in this proposal are proteins which are difficult to study by experimental techniques i.e. disordered proteins, transmembrane proteins and large complexes. The first objective will be to explore key developments in the underpinning algorithms, tackling both the issue of needing very large numbers of homologous sequences and also the downstream 3-D embedding to produce viable models. The second objective will involve experimental work with a collaborator where the idea that de novo protein design techniques might be exploited to artificially expand the set of available sequences for a given proto-family will be explored. The third objective will focus specifically on transmembrane protein modelling, where covariation-based approaches have proven to be highly effective. Here the goal will be to extend our existing FILM3 method to encompass both beta-barrel type TM proteins, but also to try to handle the issue of homomultimers, which is a critical aspect of TM protein modelling as so many families are known to adopt higher orders of structure than the fold level alone. Finally, applications of covariation analysis to probing multiple conformations of disordered proteins will be developed, with a specific focus on interactions of disordered proteins with DNA and RNA.

SummaryGenomic DNA represents the blueprint of life: it instructs solutions to challenges during life cycles of organisms. Curiously DNA in higher organisms is mostly non-protein coding (e.g. 97% in human). The popular “junk-DNA” hypothesis postulates that this non-coding DNA is non-functional. However, high-throughput transcriptomics indicates that this may be an over-simplification as most non-coding DNA is transcribed. This pervasive transcription yields two molecular events that may be functional: 1.) resulting long non-coding RNA (lncRNA) molecules, and 2.) the act of pervasive transcription itself. Whereas lncRNA sequences and functions differ on a case-by-case basis, RNA polymerase II (Pol II) transcribes most lncRNA. Pol II activity leaves molecular marks that specify transcription stages. The profiles of stage-specific activities instruct separation and fidelity of transcription units (genomic punctuation). Pervasive transcription affects genomic punctuation: upstream lncRNA transcription over gene promoters can repress downstream gene expression, also referred to as tandem Transcriptional Interference (tTI). Even though tTI was first reported decades ago a systematic characterization of tTI is lacking. Guided by my expertise in lncRNA transcription I recently identified the genetic material to dissect tTI in plants as an independent group leader. My planned research promises to reveal the genetic architecture and the molecular hallmarks defining tTI in higher organisms. Environmental lncRNA transcription variability may trigger tTI to promote organismal responses to changing conditions. We will address the roles of tTI in plant cold response to test this hypothesis. I anticipate our findings to inform on the fraction of pervasive transcription engaging in tTI. My proposal promises to advance our understanding of genomes by reconciling how the transcription of variable non-coding DNA sequences can elicit equivalent functions.

Genomic DNA represents the blueprint of life: it instructs solutions to challenges during life cycles of organisms. Curiously DNA in higher organisms is mostly non-protein coding (e.g. 97% in human). The popular “junk-DNA” hypothesis postulates that this non-coding DNA is non-functional. However, high-throughput transcriptomics indicates that this may be an over-simplification as most non-coding DNA is transcribed. This pervasive transcription yields two molecular events that may be functional: 1.) resulting long non-coding RNA (lncRNA) molecules, and 2.) the act of pervasive transcription itself. Whereas lncRNA sequences and functions differ on a case-by-case basis, RNA polymerase II (Pol II) transcribes most lncRNA. Pol II activity leaves molecular marks that specify transcription stages. The profiles of stage-specific activities instruct separation and fidelity of transcription units (genomic punctuation). Pervasive transcription affects genomic punctuation: upstream lncRNA transcription over gene promoters can repress downstream gene expression, also referred to as tandem Transcriptional Interference (tTI). Even though tTI was first reported decades ago a systematic characterization of tTI is lacking. Guided by my expertise in lncRNA transcription I recently identified the genetic material to dissect tTI in plants as an independent group leader. My planned research promises to reveal the genetic architecture and the molecular hallmarks defining tTI in higher organisms. Environmental lncRNA transcription variability may trigger tTI to promote organismal responses to changing conditions. We will address the roles of tTI in plant cold response to test this hypothesis. I anticipate our findings to inform on the fraction of pervasive transcription engaging in tTI. My proposal promises to advance our understanding of genomes by reconciling how the transcription of variable non-coding DNA sequences can elicit equivalent functions.

SummaryRNAi refers to the ability of small RNAs to silence expression of homologous sequences. A surprising link between epigenetics and RNAi was discovered more than a decade ago, and I was fortunate enough to be involved in this exciting field of research from the beginning. It is now well established that endogenous small RNAs have a direct impact on the genome in various organisms. Yet, the initiation of chromatin modifications in trans by exogenously introduced small RNAs has been inherently difficult to achieve in all eukaryotic cells. This has sparked controversy about the importance and conservation of RNAi-mediated epigenome regulation and hampered systematic mechanistic dissection of this phenomenon.
Using fission yeast, we have discovered a counter-acting mechanism that impedes small RNA-directed formation of heterochromatin and constitutes the foundation of this proposal. Our goal is to close several knowledge gaps and test the intriguing possibility that the suppressive mechanism we discovered is conserved in mammalian cells. We will employ yeast and embryonic stem cells and use cutting-edge technologies (i.e., chemical mutagenesis combined with whole-genome sequencing, genome editing with engineered nucleases, and single-cell RNA sequencing) to address fundamental, as yet unanswered questions.
My proposal consists of four major aims. In aim 1, I propose to use proteomics approaches and to perform yeast genetic screens to define additional pathway components and regulatory factors. Aim 2 builds on our ability to finally trigger de novo formation of heterochromatin by synthetic siRNAs acting in trans, addressing many of the outstanding mechanistic questions that could not be addressed in the past. In Aims 3 and 4, experiments conducted in yeast and mouse cells will elucidate missing fragments critical to our understanding of the conserved principles behind RNAi-mediated epigenetic gene regulation.

RNAi refers to the ability of small RNAs to silence expression of homologous sequences. A surprising link between epigenetics and RNAi was discovered more than a decade ago, and I was fortunate enough to be involved in this exciting field of research from the beginning. It is now well established that endogenous small RNAs have a direct impact on the genome in various organisms. Yet, the initiation of chromatin modifications in trans by exogenously introduced small RNAs has been inherently difficult to achieve in all eukaryotic cells. This has sparked controversy about the importance and conservation of RNAi-mediated epigenome regulation and hampered systematic mechanistic dissection of this phenomenon.
Using fission yeast, we have discovered a counter-acting mechanism that impedes small RNA-directed formation of heterochromatin and constitutes the foundation of this proposal. Our goal is to close several knowledge gaps and test the intriguing possibility that the suppressive mechanism we discovered is conserved in mammalian cells. We will employ yeast and embryonic stem cells and use cutting-edge technologies (i.e., chemical mutagenesis combined with whole-genome sequencing, genome editing with engineered nucleases, and single-cell RNA sequencing) to address fundamental, as yet unanswered questions.
My proposal consists of four major aims. In aim 1, I propose to use proteomics approaches and to perform yeast genetic screens to define additional pathway components and regulatory factors. Aim 2 builds on our ability to finally trigger de novo formation of heterochromatin by synthetic siRNAs acting in trans, addressing many of the outstanding mechanistic questions that could not be addressed in the past. In Aims 3 and 4, experiments conducted in yeast and mouse cells will elucidate missing fragments critical to our understanding of the conserved principles behind RNAi-mediated epigenetic gene regulation.

Max ERC Funding

1 998 557 €

Duration

Start date: 2017-01-01, End date: 2021-12-31

Project acronymRevolution

ProjectRegulation and Evolution of C4 photosynthesis

Researcher (PI)Julian Michael HIBBERD

Host Institution (HI)THE CHANCELLOR MASTERS AND SCHOLARS OF THE UNIVERSITY OF CAMBRIDGE

Call DetailsAdvanced Grant (AdG), LS2, ERC-2015-AdG

SummaryLife is dependent on sugars made during photosynthesis. When plants colonized land ~450 million years ago they used a photosynthetic system known as the C3 pathway that still operates in the majority of species today. However, from ~30 million years ago over sixty plant lineages evolved a version of photosynthesis known as the C4 pathway that increases CO2 fixation efficiency by about 50%. C4 species such as maize and sorghum are now the most productive on the planet and achieve this by compartmentalizing gene expression between cell-types.
As with other complex biological systems made up of multiple distinct cell-types, it has not been possible to understand how photosynthesis genes are regulated in specific cell-types of C4 leaves. In contrast to strategies being used by other groups, I propose to discover how specific cell-types of ancestral C3 leaves regulate gene expression, and then to use this information to determine how C4 photosynthesis operates. To achieve this, state-of-the-art approaches used on whole tissues will be adapted to study individual cell-types.
Revolution will test the hypothesis that cell-specific gene expression in C4 leaves is mediated by pre-existing regulatory networks found in C3 species. Intracellular mechanisms regulating photosynthesis genes in ancestral C3 but also derived C4 leaves will be identified. In C3 leaves I wish to understand how some cell-types express photosynthesis genes whilst others remain photosynthetically repressed. In C4 leaves I wish to discover the extent to which cell-specific expression is based upon pre-existing regulatory networks in the C3 leaf.
Revolution will therefore generate information of broad relevance to understanding gene expression in eukaryotes, and provide insight into mechanisms underpinning one of the major evolutionary transitions since plants moved to land.

Life is dependent on sugars made during photosynthesis. When plants colonized land ~450 million years ago they used a photosynthetic system known as the C3 pathway that still operates in the majority of species today. However, from ~30 million years ago over sixty plant lineages evolved a version of photosynthesis known as the C4 pathway that increases CO2 fixation efficiency by about 50%. C4 species such as maize and sorghum are now the most productive on the planet and achieve this by compartmentalizing gene expression between cell-types.
As with other complex biological systems made up of multiple distinct cell-types, it has not been possible to understand how photosynthesis genes are regulated in specific cell-types of C4 leaves. In contrast to strategies being used by other groups, I propose to discover how specific cell-types of ancestral C3 leaves regulate gene expression, and then to use this information to determine how C4 photosynthesis operates. To achieve this, state-of-the-art approaches used on whole tissues will be adapted to study individual cell-types.
Revolution will test the hypothesis that cell-specific gene expression in C4 leaves is mediated by pre-existing regulatory networks found in C3 species. Intracellular mechanisms regulating photosynthesis genes in ancestral C3 but also derived C4 leaves will be identified. In C3 leaves I wish to understand how some cell-types express photosynthesis genes whilst others remain photosynthetically repressed. In C4 leaves I wish to discover the extent to which cell-specific expression is based upon pre-existing regulatory networks in the C3 leaf.
Revolution will therefore generate information of broad relevance to understanding gene expression in eukaryotes, and provide insight into mechanisms underpinning one of the major evolutionary transitions since plants moved to land.

Max ERC Funding

2 496 521 €

Duration

Start date: 2016-09-01, End date: 2021-08-31

Project acronymRNAEPIGEN

ProjectMechanisms of epigenetic inheritance by short RNAs

Researcher (PI)Germano Cecere

Host Institution (HI)INSTITUT PASTEUR

Call DetailsStarting Grant (StG), LS2, ERC-2015-STG

SummaryEpigenetic mechanisms are considered to be central to the development of multicellular organisms made of different cell types, all having identical genomes. Similarly, they may explain how genetically identical organisms are capable of adapting to distinct environmental conditions. Yet, the molecular mechanisms regulating how epigenetic traits can be inherited during cell division or across generations are not fully understood. Using the nematode Caenorhabditis elegans, we have recently revealed how the nuclear Argonaute protein CSR-1 and its associated short RNAs participate in global transcriptional regulation and chromatin organization. This unprecedented observation opened up a new class of molecular mechanisms by which Argonaute proteins and their bound short RNAs may actively contribute to epigenetic inheritance in animals.
This research proposal focuses on the characterization of short-RNA-based mechanisms of epigenetic inheritance during animal development and upon environmental changes. Using C. elegans as an animal model system, we plan to integrate genetic, biochemical, and molecular biology tools with high-throughput genomic and proteomic approaches to dissect (i) the molecular mechanism by which CSR-1-bound short RNAs regulate transcription, (ii) test their ability in propagating the memory of actively transcribed genomic regions during early embryonic development, and (iii) characterize their role in propagating the memory of stress responses across generations to facilitate the adaptation of animals to environmental changes.
Given the association of nuclear Argonaute proteins with transcriptionally active loci in metazoans, we anticipate that similar CSR-1-like epigenetic functions are also conserved in humans. Therefore, our research has the potential to significantly advance our understanding of the molecular mechanisms underlying epigenetic inheritance and reveals their impact on animal development and adaptation to changing environments.

Epigenetic mechanisms are considered to be central to the development of multicellular organisms made of different cell types, all having identical genomes. Similarly, they may explain how genetically identical organisms are capable of adapting to distinct environmental conditions. Yet, the molecular mechanisms regulating how epigenetic traits can be inherited during cell division or across generations are not fully understood. Using the nematode Caenorhabditis elegans, we have recently revealed how the nuclear Argonaute protein CSR-1 and its associated short RNAs participate in global transcriptional regulation and chromatin organization. This unprecedented observation opened up a new class of molecular mechanisms by which Argonaute proteins and their bound short RNAs may actively contribute to epigenetic inheritance in animals.
This research proposal focuses on the characterization of short-RNA-based mechanisms of epigenetic inheritance during animal development and upon environmental changes. Using C. elegans as an animal model system, we plan to integrate genetic, biochemical, and molecular biology tools with high-throughput genomic and proteomic approaches to dissect (i) the molecular mechanism by which CSR-1-bound short RNAs regulate transcription, (ii) test their ability in propagating the memory of actively transcribed genomic regions during early embryonic development, and (iii) characterize their role in propagating the memory of stress responses across generations to facilitate the adaptation of animals to environmental changes.
Given the association of nuclear Argonaute proteins with transcriptionally active loci in metazoans, we anticipate that similar CSR-1-like epigenetic functions are also conserved in humans. Therefore, our research has the potential to significantly advance our understanding of the molecular mechanisms underlying epigenetic inheritance and reveals their impact on animal development and adaptation to changing environments.

Max ERC Funding

1 791 250 €

Duration

Start date: 2016-02-01, End date: 2021-01-31

Project acronymSynarchiC

ProjectInvestigating the functional architecture of microbial genomes with synthetic approaches

Researcher (PI)Romain KOSZUL

Host Institution (HI)INSTITUT PASTEUR

Call DetailsConsolidator Grant (CoG), LS2, ERC-2017-COG

SummaryThe folding of eukaryotic and prokaryotic chromosomes consists of an assortment of intertwined structural features. The resulting complex networks of contacts is highly dynamic and interacts functionally with, or regulates metabolic processes ranging from gene expression to chromosome segregation. Some higher order structures involve evolutionary conserved molecular players, such as structural maintenance of chromosomes (SMC) proteins, while others depend on phylum specific proteins. The functional organization of yeast and bacteria chromosomes are actively investigated, with multiple folded structures uncovered in recent years. However, disambiguating the intermingled structures is a difficult task, limiting their functional characterization. In addition, current technologies are limited and are unable to track the genome-wide folding of duplicated sister chromatids (SC) molecules, limiting the study of genome folding during replication and mitosis.
The overall aim of the SynarchiC project is to characterize, through innovative derivatives of the chromosome conformation capture technology combined with synthetic chromosomes, the folding patterns of microbial genomes during the entire cell cycle, including those of SCs. By reverse engineering chromosomes in bacteria and yeast, we will discriminate the different layers of topological structures and their associated molecular players. We will then investigate how these 3D structures affect SC folding, individualization, and segregation. Finally, we will investigate the interplay between a pathogen and its hosts during an infectious process. How the bacteria redirects its host chromosome metabolism in stressful environment will be addressed from the perspective of genome organization and segregation. Technologies and results from SynarchiC will provide fundamental insights on the cell cycle, and should appeal broadly to scientists working on various aspects genome functional organization in any clade.

The folding of eukaryotic and prokaryotic chromosomes consists of an assortment of intertwined structural features. The resulting complex networks of contacts is highly dynamic and interacts functionally with, or regulates metabolic processes ranging from gene expression to chromosome segregation. Some higher order structures involve evolutionary conserved molecular players, such as structural maintenance of chromosomes (SMC) proteins, while others depend on phylum specific proteins. The functional organization of yeast and bacteria chromosomes are actively investigated, with multiple folded structures uncovered in recent years. However, disambiguating the intermingled structures is a difficult task, limiting their functional characterization. In addition, current technologies are limited and are unable to track the genome-wide folding of duplicated sister chromatids (SC) molecules, limiting the study of genome folding during replication and mitosis.
The overall aim of the SynarchiC project is to characterize, through innovative derivatives of the chromosome conformation capture technology combined with synthetic chromosomes, the folding patterns of microbial genomes during the entire cell cycle, including those of SCs. By reverse engineering chromosomes in bacteria and yeast, we will discriminate the different layers of topological structures and their associated molecular players. We will then investigate how these 3D structures affect SC folding, individualization, and segregation. Finally, we will investigate the interplay between a pathogen and its hosts during an infectious process. How the bacteria redirects its host chromosome metabolism in stressful environment will be addressed from the perspective of genome organization and segregation. Technologies and results from SynarchiC will provide fundamental insights on the cell cycle, and should appeal broadly to scientists working on various aspects genome functional organization in any clade.

Max ERC Funding

1 995 557 €

Duration

Start date: 2018-04-01, End date: 2023-03-31

Project acronymSynthHotSpot

ProjectSynthesizing Meiotic Crossover Hotspots in Arabidopsis

Researcher (PI)Ian Robert Henderson

Host Institution (HI)THE CHANCELLOR MASTERS AND SCHOLARS OF THE UNIVERSITY OF CAMBRIDGE

Call DetailsConsolidator Grant (CoG), LS2, ERC-2015-CoG

SummaryThe majority of eukaryotes reproduce sexually via meiosis. During meiosis homologous chromosomes pair and undergo reciprocal genetic exchange termed crossover. Meiotic recombination is a major evolutionary force and has a profound effect on patterns of genetic diversity in sexual species. Crossovers distributions are highly non-random and are typically focused in narrow hotspots. Study of hotspots throughout eukaryotes has revealed combinations of genetic and epigenetic factors that contribute to their distributions. In this proposal we will use the extensive genetics and genomics tools available in Arabidopsis to comprehensively dissect hotspot patterning. The strategic aim of the proposal is to use this knowledge to direct de novo hotspots to loci of choice. In the first aim we will use functional genomics to profile the chromosomal distributions of key recombination proteins and test the role of chromatin and higher-order structures in driving these patterns. In the second aim we will study individual hotspots at the fine-scale, to the resolution of individual polymorphisms, using amplification and sequencing of recombinant molecules from gamete DNA. To test genetic versus epigenetic control of hotspots we will use genome-editing to delete hotspot-associated CTT-repeat DNA sequence motifs, in addition to directing DNA methylation in order to epigenetically silence recombination. In the final aim we will use our combined knowledge of hotspot control to implement genome-editing technologies (TALENs & CRISPR-Cas9) during meiosis. This will allow us to rationally control hotspot locations, which will be definitive proof that our models for recombination control are correct. This technology will also accelerate breeding and genome-engineering of our most important crops, where recombination can be limiting. Finally, mapping hotspots will allow us to better understand patterns of natural genetic diversity, including detecting the signatures of selection.

The majority of eukaryotes reproduce sexually via meiosis. During meiosis homologous chromosomes pair and undergo reciprocal genetic exchange termed crossover. Meiotic recombination is a major evolutionary force and has a profound effect on patterns of genetic diversity in sexual species. Crossovers distributions are highly non-random and are typically focused in narrow hotspots. Study of hotspots throughout eukaryotes has revealed combinations of genetic and epigenetic factors that contribute to their distributions. In this proposal we will use the extensive genetics and genomics tools available in Arabidopsis to comprehensively dissect hotspot patterning. The strategic aim of the proposal is to use this knowledge to direct de novo hotspots to loci of choice. In the first aim we will use functional genomics to profile the chromosomal distributions of key recombination proteins and test the role of chromatin and higher-order structures in driving these patterns. In the second aim we will study individual hotspots at the fine-scale, to the resolution of individual polymorphisms, using amplification and sequencing of recombinant molecules from gamete DNA. To test genetic versus epigenetic control of hotspots we will use genome-editing to delete hotspot-associated CTT-repeat DNA sequence motifs, in addition to directing DNA methylation in order to epigenetically silence recombination. In the final aim we will use our combined knowledge of hotspot control to implement genome-editing technologies (TALENs & CRISPR-Cas9) during meiosis. This will allow us to rationally control hotspot locations, which will be definitive proof that our models for recombination control are correct. This technology will also accelerate breeding and genome-engineering of our most important crops, where recombination can be limiting. Finally, mapping hotspots will allow us to better understand patterns of natural genetic diversity, including detecting the signatures of selection.

SummaryMass spectrometry-based proteomics and next generation DNA sequencing emerged as two powerful and complementary technologies in biology. I was the first to integrate these technologies in the area of epigenetics to identify and functionally characterize proteins that interact with post-translational modifications on histones and (hydroxy)methylated DNA (so-called chromatin ‘readers’). My pioneering work revealed that an intricate networks of transcription factors, chromatin modifications and chromatin readers orchestrate dynamic gene expression programs during embryonic stem cell differentiation. The next big challenge is to understand the molecular mechanisms, which help to control maintenance and differentiation of adult stem cells as an integral part of an organ. Intestinal organoid cultures recently emerged as a paradigm to study adult stem cell maintenance and differentiation. These ‘miniguts’ can be cultured in vitro and contain all the different cell types that are present in the mouse small intestinal epithelium. Recently it was shown that small-molecule driven perturbations can be used to obtain organoids which are strongly enriched for specific intestinal cell types. This system thus provides a perfect opportunity to study for the first time and in a controlled manner, adult stem cell maintenance and (de)differentiation. Using small molecule-driven perturbations and a unique combination of ‘omics’ technologies, which are embedded in my department, I will provide a systems-wide view of the molecular (epigenetic) mechanisms that orchestrate cell fate changes in intestinal organoids. This integrative approach will identify the major regulatory networks that define the remarkable cellular plasticity of the mouse small intestinal epithelium. Beyond this basic scientific goal, our work will also have profound implications for cancer research and regenerative medicine, both of which are characterized by changes in adult stem cell homeostasis.

Mass spectrometry-based proteomics and next generation DNA sequencing emerged as two powerful and complementary technologies in biology. I was the first to integrate these technologies in the area of epigenetics to identify and functionally characterize proteins that interact with post-translational modifications on histones and (hydroxy)methylated DNA (so-called chromatin ‘readers’). My pioneering work revealed that an intricate networks of transcription factors, chromatin modifications and chromatin readers orchestrate dynamic gene expression programs during embryonic stem cell differentiation. The next big challenge is to understand the molecular mechanisms, which help to control maintenance and differentiation of adult stem cells as an integral part of an organ. Intestinal organoid cultures recently emerged as a paradigm to study adult stem cell maintenance and differentiation. These ‘miniguts’ can be cultured in vitro and contain all the different cell types that are present in the mouse small intestinal epithelium. Recently it was shown that small-molecule driven perturbations can be used to obtain organoids which are strongly enriched for specific intestinal cell types. This system thus provides a perfect opportunity to study for the first time and in a controlled manner, adult stem cell maintenance and (de)differentiation. Using small molecule-driven perturbations and a unique combination of ‘omics’ technologies, which are embedded in my department, I will provide a systems-wide view of the molecular (epigenetic) mechanisms that orchestrate cell fate changes in intestinal organoids. This integrative approach will identify the major regulatory networks that define the remarkable cellular plasticity of the mouse small intestinal epithelium. Beyond this basic scientific goal, our work will also have profound implications for cancer research and regenerative medicine, both of which are characterized by changes in adult stem cell homeostasis.

Max ERC Funding

2 000 000 €

Duration

Start date: 2018-10-01, End date: 2023-09-30

Project acronymTarMyc

ProjectTargeting the Oncogenic Function of Myc in vivo

Researcher (PI)Elmar WOLF

Host Institution (HI)JULIUS-MAXIMILIANS-UNIVERSITAT WURZBURG

Call DetailsStarting Grant (StG), LS2, ERC-2017-STG

SummaryThe transcription factor Myc plays a central role in tumourigenesis but was deemed undruggable due to it being an essential protein. However, recent proof-of-principle studies in mice using a dominant negative allele of Myc demonstrated the dependency of established tumours on Myc function and showed that mice tolerated Myc inhibition to a degree that allowed tumour regression. In line with these observations my group found Myc to regulate distinct sets of genes at low, physiological and high, oncogenic levels, because promoters differ in their affinity for Myc. This notion implies the compelling possibility to specifically target the oncogenic functions of Myc.
TarMyc aims to address four key questions required to bring this new concept from bench to bedside. Firstly, TarMyc will estimate the therapeutic window of Myc inhibition in vivo by expressing shRNAs against Myc in mice with established solid tumours. Secondly, TarMyc aims to identify in vivo Myc target genes crucial for tumourigenesis. Thirdly, this proposal aims to elucidate the role of Myc’s differential promoter affinity in untransformed cells. Analysis of published gene expression datasets revealed Myc binding to low-affinity promoters during the process of tissue regeneration. Thus, by characterizing the regeneration programme induced by Myc we hope to gain further insight on the therapeutic window of Myc inhibition and assess potential side-effects in a Myc-targeting anticancer therapy. Fourthly, we aim to develop strategies to interfere with the oncogenic functions of Myc by (i) developing a novel class of drugs that reduce Myc’s cellular concentrations, and (ii) by testing the therapeutic potential of Myc target genes by inhibiting their function in tumour models.
Taken together, TarMyc takes on the challenge of inhibiting the oncogenic functions of Myc in a highly multidisciplinary approach using state-of-the-art molecular biology, advanced tumour models and new concepts in drug development.

The transcription factor Myc plays a central role in tumourigenesis but was deemed undruggable due to it being an essential protein. However, recent proof-of-principle studies in mice using a dominant negative allele of Myc demonstrated the dependency of established tumours on Myc function and showed that mice tolerated Myc inhibition to a degree that allowed tumour regression. In line with these observations my group found Myc to regulate distinct sets of genes at low, physiological and high, oncogenic levels, because promoters differ in their affinity for Myc. This notion implies the compelling possibility to specifically target the oncogenic functions of Myc.
TarMyc aims to address four key questions required to bring this new concept from bench to bedside. Firstly, TarMyc will estimate the therapeutic window of Myc inhibition in vivo by expressing shRNAs against Myc in mice with established solid tumours. Secondly, TarMyc aims to identify in vivo Myc target genes crucial for tumourigenesis. Thirdly, this proposal aims to elucidate the role of Myc’s differential promoter affinity in untransformed cells. Analysis of published gene expression datasets revealed Myc binding to low-affinity promoters during the process of tissue regeneration. Thus, by characterizing the regeneration programme induced by Myc we hope to gain further insight on the therapeutic window of Myc inhibition and assess potential side-effects in a Myc-targeting anticancer therapy. Fourthly, we aim to develop strategies to interfere with the oncogenic functions of Myc by (i) developing a novel class of drugs that reduce Myc’s cellular concentrations, and (ii) by testing the therapeutic potential of Myc target genes by inhibiting their function in tumour models.
Taken together, TarMyc takes on the challenge of inhibiting the oncogenic functions of Myc in a highly multidisciplinary approach using state-of-the-art molecular biology, advanced tumour models and new concepts in drug development.

Max ERC Funding

1 497 905 €

Duration

Start date: 2018-03-01, End date: 2023-02-28

Project acronymTranslationRegCode

ProjectCracking the Translation Regulatory Code

Researcher (PI)Reut Gitit Shalgi

Host Institution (HI)TECHNION - ISRAEL INSTITUTE OF TECHNOLOGY

Call DetailsStarting Grant (StG), LS2, ERC-2015-STG

SummaryOrganisms across all kingdoms share several systems that are essential to life, one of the most central being protein synthesis. Living in a continuously changing environment, cells need to constantly respond to various environmental cues and change their protein landscape. In extreme cases, cells globally shut down protein synthesis and upregulate stress-protective proteins.
Mechanisms of translational repression or selective enhancement of stress-induced proteins have been characterized, but their effects were demonstrated on an individual mRNA basis. Which target mRNAs are translationally regulated in response to different environmental cues, and what are the cis-regulatory elements involved, largely remain as open questions. Using ribosome footprint profiling, I recently discovered a novel mode of translational control in stress, underscoring the potential of new technologies to uncover novel regulatory mechanisms. But while transcription cis-regulatory elements have been thoroughly mapped in the past decade, and splicing regulatory elements are accumulating, the identification of translation cis-regulatory elements is lagging behind.
Here I propose to crack the mammalian translation regulatory code, and close this long-standing gap. I present a novel interdisciplinary framework to comprehensively identify translation cis-regulatory elements, and map their mRNAs targets in a variety of cellular perturbations. Importantly, we plan to explore mechanisms underlying novel cis-regulatory elements, and create the first genome-wide functionally annotated translation regulatory code.
The translation regulatory code will map targets of existing mechanisms and shed light on newly identified pathways that play a role in stress-induced translational control. The proposed project is an imperative stepping stone to understanding translational regulation by cis-regulatory elements, opening new avenues in the functional genomics research of translational control.

Organisms across all kingdoms share several systems that are essential to life, one of the most central being protein synthesis. Living in a continuously changing environment, cells need to constantly respond to various environmental cues and change their protein landscape. In extreme cases, cells globally shut down protein synthesis and upregulate stress-protective proteins.
Mechanisms of translational repression or selective enhancement of stress-induced proteins have been characterized, but their effects were demonstrated on an individual mRNA basis. Which target mRNAs are translationally regulated in response to different environmental cues, and what are the cis-regulatory elements involved, largely remain as open questions. Using ribosome footprint profiling, I recently discovered a novel mode of translational control in stress, underscoring the potential of new technologies to uncover novel regulatory mechanisms. But while transcription cis-regulatory elements have been thoroughly mapped in the past decade, and splicing regulatory elements are accumulating, the identification of translation cis-regulatory elements is lagging behind.
Here I propose to crack the mammalian translation regulatory code, and close this long-standing gap. I present a novel interdisciplinary framework to comprehensively identify translation cis-regulatory elements, and map their mRNAs targets in a variety of cellular perturbations. Importantly, we plan to explore mechanisms underlying novel cis-regulatory elements, and create the first genome-wide functionally annotated translation regulatory code.
The translation regulatory code will map targets of existing mechanisms and shed light on newly identified pathways that play a role in stress-induced translational control. The proposed project is an imperative stepping stone to understanding translational regulation by cis-regulatory elements, opening new avenues in the functional genomics research of translational control.

Max ERC Funding

1 587 500 €

Duration

Start date: 2016-03-01, End date: 2021-02-28

Project acronymTRANSPOS-X

ProjectTransposable elements, their controllers and the genesis of human-specific transcriptional networks

Researcher (PI)Didier Trono

Host Institution (HI)ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE

Call DetailsAdvanced Grant (AdG), LS2, ERC-2015-AdG

SummaryTransposable elements (TEs) account for more than two thirds of the human genome. They can inactivate
genes, provide novel coding functions, sprinkle chromosomes with recombination-prone repetitive
sequences, and modulate cellular gene expression through a wide variety of transcriptional and
posttranscriptional influences. As a consequence, TEs are considered as essential motors of evolution yet
they are occasionally associated with disease, causing about one hundred Mendelian disorders and possibly
contributing to several human cancers. As expected for such genomic threats, TEs are subjected to tight
epigenetic control imposed from the very first days of embryogenesis, in part owing to their recognition by
sequence-specific RNA- and protein-based repressors. It is generally considered that the evolutionary
selection of these TE controllers reflects a simple host-pathogen arms race, and that their action results in the
early and permanent silencing of their targets. We have recently uncovered new evolutionary evidence and
obtained genomic and functional data that invalidate this dual assumption, and suggest instead that
transposable elements and their epigenetic controllers establish species-specific transcriptional networks that
play critical roles in human development and physiology. The general objective of the present proposal is to
explore the breadth of this phenomenon, to decipher its mechanisms, to unveil its functional implications,
and to probe how this knowledge could be exploited for basic research, biotechnology and clinical medicine.

Transposable elements (TEs) account for more than two thirds of the human genome. They can inactivate
genes, provide novel coding functions, sprinkle chromosomes with recombination-prone repetitive
sequences, and modulate cellular gene expression through a wide variety of transcriptional and
posttranscriptional influences. As a consequence, TEs are considered as essential motors of evolution yet
they are occasionally associated with disease, causing about one hundred Mendelian disorders and possibly
contributing to several human cancers. As expected for such genomic threats, TEs are subjected to tight
epigenetic control imposed from the very first days of embryogenesis, in part owing to their recognition by
sequence-specific RNA- and protein-based repressors. It is generally considered that the evolutionary
selection of these TE controllers reflects a simple host-pathogen arms race, and that their action results in the
early and permanent silencing of their targets. We have recently uncovered new evolutionary evidence and
obtained genomic and functional data that invalidate this dual assumption, and suggest instead that
transposable elements and their epigenetic controllers establish species-specific transcriptional networks that
play critical roles in human development and physiology. The general objective of the present proposal is to
explore the breadth of this phenomenon, to decipher its mechanisms, to unveil its functional implications,
and to probe how this knowledge could be exploited for basic research, biotechnology and clinical medicine.

Max ERC Funding

2 500 000 €

Duration

Start date: 2017-01-01, End date: 2021-12-31

Project acronymTransposonsReprogram

ProjectHow retrotransposons remodel the genome during early development and reprogramming

Researcher (PI)HELEN MARY Rowe

Host Institution (HI)UNIVERSITY COLLEGE LONDON

Call DetailsStarting Grant (StG), LS2, ERC-2015-STG

SummaryRetrotransposons (RTNs) are ancient viruses that have stably integrated themselves into mammalian genomes and they now occupy around half of the human or mouse genome. These mobile genetic elements that have coevolved with us drive evolution by creating new genes and plasticity of genomes. Exciting data including ours has shown that even RTNs that are no longer active retain enhancer, promoter or repressor sequences that regulate developmental genes, through largely uncharacterized transcription factors. We have employed CRISPR/Cas9 gene disruption to determine that Zfp37 and Zfp819 bind to and regulate RTNs in mouse embryonic stem cells (ESCs). Identification of these zinc finger proteins (ZFPs) now allows us to ask new questions about how RTNs have been co-opted to orchestrate gene circuits in vitro and in vivo. Both these factors have already been implicated to play a role in reprogramming or genome integrity.
We hypothesize that RTNs have been co-opted to remodel the genome by acting as structural platforms that recruit transcription factors like Zfp37 and Zfp819. We will test this hypothesis assessing the role of RTNs and these two ZFPs in three dynamic contexts where the genome is remodelled. These are in ESC differentiation to neurons, in reprogramming and in early mouse development, three scenarios where RTNs have been documented to become expressed and serve an unknown function.
This work will exploit mouse development to unravel the mechanism of how RTNs remodel the genome. It will help us to understand how ZFPs can be engaged to reprogram cells and in stem-cell therapies, and will explain more broadly how RTNs, which dominate our genomes, control cell fate.

Retrotransposons (RTNs) are ancient viruses that have stably integrated themselves into mammalian genomes and they now occupy around half of the human or mouse genome. These mobile genetic elements that have coevolved with us drive evolution by creating new genes and plasticity of genomes. Exciting data including ours has shown that even RTNs that are no longer active retain enhancer, promoter or repressor sequences that regulate developmental genes, through largely uncharacterized transcription factors. We have employed CRISPR/Cas9 gene disruption to determine that Zfp37 and Zfp819 bind to and regulate RTNs in mouse embryonic stem cells (ESCs). Identification of these zinc finger proteins (ZFPs) now allows us to ask new questions about how RTNs have been co-opted to orchestrate gene circuits in vitro and in vivo. Both these factors have already been implicated to play a role in reprogramming or genome integrity.
We hypothesize that RTNs have been co-opted to remodel the genome by acting as structural platforms that recruit transcription factors like Zfp37 and Zfp819. We will test this hypothesis assessing the role of RTNs and these two ZFPs in three dynamic contexts where the genome is remodelled. These are in ESC differentiation to neurons, in reprogramming and in early mouse development, three scenarios where RTNs have been documented to become expressed and serve an unknown function.
This work will exploit mouse development to unravel the mechanism of how RTNs remodel the genome. It will help us to understand how ZFPs can be engaged to reprogram cells and in stem-cell therapies, and will explain more broadly how RTNs, which dominate our genomes, control cell fate.

Max ERC Funding

1 499 055 €

Duration

Start date: 2016-05-01, End date: 2021-12-31

Project acronymUbl-Code

ProjectRevealing the ubiquitin and ubiquitin-like modification landscape in health and disease

Researcher (PI)Yifat Haya Merbl

Host Institution (HI)WEIZMANN INSTITUTE OF SCIENCE LTD

Call DetailsStarting Grant (StG), LS2, ERC-2015-STG

SummaryPost-translational modifications (PTMs) of proteins are a major tool that the cell uses to monitor events and initiate appropriate responses. While a protein is defined by its backbone of amino acid sequence, its function is often determined by PTMs, which specify stability, activity, or cellular localization. Among PTMs, ubiquitin and ubiquitin-like (Ubl) modifications were shown to regulate a variety of fundamental cellular processes such as cell division and differentiation. Aberrations in these pathways have been implicated in the pathogenesis of cancer. Over the past decade high-throughput genomic and transcriptional analyses have profoundly broadened our understanding of the processes underlying cancer development and progression. Yet, proteomic analyses and the PTM landscape in cancer, remained relatively unexplored.
Our goal is to decipher molecular mechanisms of Ubl regulation in cancer. We will utilize the PTM profiling technology that I developed and further develop it to allow for subsequent MS analysis. Together with cutting-edge genomic, imaging and proteomic technologies, we will analyze novel aspects of PTM regulation at the level of the enzymatic machinery, the substrates and the downstream cellular network. We will rely on ample in-vitro and in-vivo characterization of Ubl conjugation to:a. Elucidate the regulatory principles of substrate specificity and recognition. b. Understand signalling dynamics in the ubiquitin system. c. Reveal how aberrations in these pathways may lead to diseases such as cancer. Identifying both the Ubl modifying enzymes and the modified substrates will form the basis for deciphering the molecular pathways in which they operate in the cell and the principles of their dynamic regulation. Revealing the PTM regulatory code presents a unique opportunity for the development of novel therapeutics. More broadly, our approaches may provide a new paradigm for addressing other complex biological questions involving PTM regulation.

Post-translational modifications (PTMs) of proteins are a major tool that the cell uses to monitor events and initiate appropriate responses. While a protein is defined by its backbone of amino acid sequence, its function is often determined by PTMs, which specify stability, activity, or cellular localization. Among PTMs, ubiquitin and ubiquitin-like (Ubl) modifications were shown to regulate a variety of fundamental cellular processes such as cell division and differentiation. Aberrations in these pathways have been implicated in the pathogenesis of cancer. Over the past decade high-throughput genomic and transcriptional analyses have profoundly broadened our understanding of the processes underlying cancer development and progression. Yet, proteomic analyses and the PTM landscape in cancer, remained relatively unexplored.
Our goal is to decipher molecular mechanisms of Ubl regulation in cancer. We will utilize the PTM profiling technology that I developed and further develop it to allow for subsequent MS analysis. Together with cutting-edge genomic, imaging and proteomic technologies, we will analyze novel aspects of PTM regulation at the level of the enzymatic machinery, the substrates and the downstream cellular network. We will rely on ample in-vitro and in-vivo characterization of Ubl conjugation to:a. Elucidate the regulatory principles of substrate specificity and recognition. b. Understand signalling dynamics in the ubiquitin system. c. Reveal how aberrations in these pathways may lead to diseases such as cancer. Identifying both the Ubl modifying enzymes and the modified substrates will form the basis for deciphering the molecular pathways in which they operate in the cell and the principles of their dynamic regulation. Revealing the PTM regulatory code presents a unique opportunity for the development of novel therapeutics. More broadly, our approaches may provide a new paradigm for addressing other complex biological questions involving PTM regulation.

Max ERC Funding

1 500 000 €

Duration

Start date: 2016-05-01, End date: 2021-04-30

Project acronymUNICODE

ProjectEvolution and Impact of Heterochromatin on a Young Drosophila Y chromosome

Researcher (PI)Qi Zhou

Host Institution (HI)UNIVERSITAT WIEN

Call DetailsStarting Grant (StG), LS2, ERC-2015-STG

SummaryThe transition from euchromatin to heterochromatin is a fundamental process that particularly reshaped the epigenomic landscape of Y chromosome. Its definitive genomic underpinning and broad functional impact are still unclear, as heterochromatin (e.g., that of human Y) is usually too repetitive to study. I have previously demonstrated that, the young Y (‘neo-Y’) chromosome of Drosophila miranda has just initiated such a transition, thus is a powerful model to unveil the evolution, regulation and functional interaction of heterochromatin. I showed that this neo-Y still harbours over 1800 genes, and only 20-50% of the sequences are transposable elements (TE). Over five years, I aim to: 1) precisely resolve the structure and insertion sites of TEs as a pre-requisite for studying heterochromatin, by combining state-of-art sequencing and bioinformatic techniques. 2) I will reveal the de novo heterochromatin formation triggered by TE insertions or the heterochromatin/euchromatin boundary shifts on the neo-Y, by comparing the binding profiles of histone modification hallmarks and insulator proteins of D. miranda to its sibling species D. pseudoobscura, which lacks the neo-Y. Such epigenomic changes have likely driven the exaptation or innovation of small RNA pathways that govern the TE mobility. 3) I will then identify the responsible small RNAs and their encoding loci, which are expected to have newly emerged or differentially expressed in D. miranda relative to D. pseudoobscura. 4) Finally, I will develop CRISPR/Cas9 in D. miranda to manipulate the expression of TEs encoding such small RNAs on the neo-Y, in order to scrutinize how TE/heterochromatin evolution on the Y would impact the chromatin landscape of the entire host genome. The combined aim of this multidisciplinary project is to generate a framework for understanding the basic mechanisms of how heterochromatin evolves; and open a new avenue toward the discovery of Y chromosome function beyond male determination.

The transition from euchromatin to heterochromatin is a fundamental process that particularly reshaped the epigenomic landscape of Y chromosome. Its definitive genomic underpinning and broad functional impact are still unclear, as heterochromatin (e.g., that of human Y) is usually too repetitive to study. I have previously demonstrated that, the young Y (‘neo-Y’) chromosome of Drosophila miranda has just initiated such a transition, thus is a powerful model to unveil the evolution, regulation and functional interaction of heterochromatin. I showed that this neo-Y still harbours over 1800 genes, and only 20-50% of the sequences are transposable elements (TE). Over five years, I aim to: 1) precisely resolve the structure and insertion sites of TEs as a pre-requisite for studying heterochromatin, by combining state-of-art sequencing and bioinformatic techniques. 2) I will reveal the de novo heterochromatin formation triggered by TE insertions or the heterochromatin/euchromatin boundary shifts on the neo-Y, by comparing the binding profiles of histone modification hallmarks and insulator proteins of D. miranda to its sibling species D. pseudoobscura, which lacks the neo-Y. Such epigenomic changes have likely driven the exaptation or innovation of small RNA pathways that govern the TE mobility. 3) I will then identify the responsible small RNAs and their encoding loci, which are expected to have newly emerged or differentially expressed in D. miranda relative to D. pseudoobscura. 4) Finally, I will develop CRISPR/Cas9 in D. miranda to manipulate the expression of TEs encoding such small RNAs on the neo-Y, in order to scrutinize how TE/heterochromatin evolution on the Y would impact the chromatin landscape of the entire host genome. The combined aim of this multidisciplinary project is to generate a framework for understanding the basic mechanisms of how heterochromatin evolves; and open a new avenue toward the discovery of Y chromosome function beyond male determination.

Max ERC Funding

1 971 846 €

Duration

Start date: 2016-08-01, End date: 2021-07-31

Project acronymUPRmt

ProjectThe Mitochondrial Unfolded Protein Response

Researcher (PI)Johan Henri Louise AUWERX

Host Institution (HI)ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE

Call DetailsAdvanced Grant (AdG), LS2, ERC-2017-ADG

SummaryMitochondria—organelles specialized in energy harvesting through oxidative phosphorylation (Oxphos)—critically influence metabolism, health and lifespan. Evolved from endosymbiotic proteobacteria, mitochondria retained the vestige of the bacterial genome, the mitochondrial DNA, which encodes 13 subunits of the Oxphos complexes, while the remaining ~80 Oxphos components and the rest of the mitochondrial proteome are encoded on nuclear DNA, translated in the cytoplasm and imported in the mitochondria. The control of the mitochondrial proteome by two genomes exposes these organelles to proteotoxic stress in case of an imbalance between the nuclear- and mitochondrial-encoded proteins. Upon such stress, several mitochondrial protein quality control (mtPQC) pathways, including the mitochondrial unfolded protein response (UPRmt), will sense, transmit and re-establish mitochondrial proteostasis through mitonuclear regulatory circuits. Although a robust UPRmt circuit improves health and lifespan in C. elegans, much less is known about mtPQC in vertebrates. We propose here to characterize UPRmt pathways across 3 species by: (1) mapping mammalian UPRmt genes and networks in vivo after the induction of the UPRmt in a large murine genetic reference population at 3 different times throughout life with 2 different inducers; (2) integrating these UPRmt networks with a wide set of clinical, mitochondrial, and molecular phenotypes collected throughout life to establish links between UPRmt mechanisms and health- and lifespan; (3) mechanistically validating the most important UPRmt pathways, using loss-of-function studies in cells, worms and mice; and (4) clinically translating promising UPRmt hits, using genetic association studies in human cohorts. The insight gained will mechanistically define the UPRmt networks from worms to humans and will provide the next step in translating the benefits of activating the UPRmt—initially observed in invertebrates—into targeted human therapies.

Mitochondria—organelles specialized in energy harvesting through oxidative phosphorylation (Oxphos)—critically influence metabolism, health and lifespan. Evolved from endosymbiotic proteobacteria, mitochondria retained the vestige of the bacterial genome, the mitochondrial DNA, which encodes 13 subunits of the Oxphos complexes, while the remaining ~80 Oxphos components and the rest of the mitochondrial proteome are encoded on nuclear DNA, translated in the cytoplasm and imported in the mitochondria. The control of the mitochondrial proteome by two genomes exposes these organelles to proteotoxic stress in case of an imbalance between the nuclear- and mitochondrial-encoded proteins. Upon such stress, several mitochondrial protein quality control (mtPQC) pathways, including the mitochondrial unfolded protein response (UPRmt), will sense, transmit and re-establish mitochondrial proteostasis through mitonuclear regulatory circuits. Although a robust UPRmt circuit improves health and lifespan in C. elegans, much less is known about mtPQC in vertebrates. We propose here to characterize UPRmt pathways across 3 species by: (1) mapping mammalian UPRmt genes and networks in vivo after the induction of the UPRmt in a large murine genetic reference population at 3 different times throughout life with 2 different inducers; (2) integrating these UPRmt networks with a wide set of clinical, mitochondrial, and molecular phenotypes collected throughout life to establish links between UPRmt mechanisms and health- and lifespan; (3) mechanistically validating the most important UPRmt pathways, using loss-of-function studies in cells, worms and mice; and (4) clinically translating promising UPRmt hits, using genetic association studies in human cohorts. The insight gained will mechanistically define the UPRmt networks from worms to humans and will provide the next step in translating the benefits of activating the UPRmt—initially observed in invertebrates—into targeted human therapies.

Max ERC Funding

2 500 000 €

Duration

Start date: 2018-11-01, End date: 2023-10-31

Project acronymYEASTMEMORY

ProjectMemory in biological regulatory circuits

Researcher (PI)Kevin Joan Verstrepen

Host Institution (HI)VIB

Call DetailsConsolidator Grant (CoG), LS2, ERC-2015-CoG

SummaryThe emergence of intelligence –the ability to remember and analyze data to make decisions– was a milestone in evolution. Intelligence and memory are usually associated with plastic neuronal connections in higher organisms. However, new discoveries hint that a rudimentary form of intelligence is rooted in networks that regulate gene expression in a wide range of organisms, including bacteria and yeasts. Specifically, we and others have shown that microbes show plastic behavioral responses to past experiences, such as previously available nutrients or stresses. This implies that information about the past is somehow retained and passed to next generations, where it influences cellular regulation.
The goal of this project is to use a simple eukaryotic regulatory circuit as a model to obtain a comprehensive picture of the different genes and molecular mechanisms underlying history-dependence (hysteresis) in cellular regulation. Specifically, we will study maltose (MAL) regulation in budding yeast, because this signaling pathway serves as a model for gene regulation circuits in other organisms, including humans. We will use a combination of genetic screens, live-cell microscopy in custom-built microfluidic devices, and mathematical modeling to pursue four aims:
1. To provide a comprehensive quantitative analysis of hysteresis in MAL regulation
2. To unravel the molecular mechanisms contributing to hysteresis
3. To unravel the epigenetic mechanisms allowing hysteresis to extend over several generations
4. To characterize the ecological relevance of hysteresis
This project will establish an innovative model for hysteresis and generate a genome-wide, systems-level view of how past influences can be stored in regulatory cascades to influence cellular decision-making. The results will contribute to a paradigm shift in our view of biological regulation and memory, with possible applications in fields as diverse as industrial microbiology, synthetic biology and medicine.

The emergence of intelligence –the ability to remember and analyze data to make decisions– was a milestone in evolution. Intelligence and memory are usually associated with plastic neuronal connections in higher organisms. However, new discoveries hint that a rudimentary form of intelligence is rooted in networks that regulate gene expression in a wide range of organisms, including bacteria and yeasts. Specifically, we and others have shown that microbes show plastic behavioral responses to past experiences, such as previously available nutrients or stresses. This implies that information about the past is somehow retained and passed to next generations, where it influences cellular regulation.
The goal of this project is to use a simple eukaryotic regulatory circuit as a model to obtain a comprehensive picture of the different genes and molecular mechanisms underlying history-dependence (hysteresis) in cellular regulation. Specifically, we will study maltose (MAL) regulation in budding yeast, because this signaling pathway serves as a model for gene regulation circuits in other organisms, including humans. We will use a combination of genetic screens, live-cell microscopy in custom-built microfluidic devices, and mathematical modeling to pursue four aims:
1. To provide a comprehensive quantitative analysis of hysteresis in MAL regulation
2. To unravel the molecular mechanisms contributing to hysteresis
3. To unravel the epigenetic mechanisms allowing hysteresis to extend over several generations
4. To characterize the ecological relevance of hysteresis
This project will establish an innovative model for hysteresis and generate a genome-wide, systems-level view of how past influences can be stored in regulatory cascades to influence cellular decision-making. The results will contribute to a paradigm shift in our view of biological regulation and memory, with possible applications in fields as diverse as industrial microbiology, synthetic biology and medicine.

SummarySeveral human pancreatic diseases have been characterized, being the diabetes the most common. Like others, this genetic disease is related to disrupted non-coding cis-regulatory elements (CREs) that culminate in altered gene expression. Although Genome Wide Association Studies support this hypothesis, it’s still unclear how mutations on CREs contribute to disease. The translation from the “non-coding code” to phenotype is an exciting and unexplored field that we will approach in this project with the help of the zebrafish as a suitable animal model. We aim to uncover the implications of the disruption of pancreas CREs and how they contribute to diabetes in vivo. For this we will study transcriptional regulation of genes in zebrafish. The similarities between zebrafish and mammal pancreas and the evolutionary conservation of pancreas transcription factors (TF) make it an excellent model to approach and study this disease. In this project we will characterize the zebrafish insulin producing beta-cell regulome, by determining the active CREs in this cell type and their bound TFs. Then we will compare this information with a similar dataset recently available for human beta-cells, to define functional orthologs in these species. Selected CREs will be tested by in vivo gene reporter assays in zebrafish, focusing on those functionally equivalent to human CREs where risk alleles have been associated with diabetes or those regulating genes involved in diabetes. Later these CREs will be mutated in the zebrafish genome to validate their contribution to diabetes. Finally we will translate this to predict new human disease-associated CREs by focusing on the regulatory landscape of diabetes-associated genes, without the need of having countless patients to uncover them. With this project we will create a model system that will allow the identification of new diabetes-associated CREs, which might have a great impact in clinical management of this epidemic disease.

Several human pancreatic diseases have been characterized, being the diabetes the most common. Like others, this genetic disease is related to disrupted non-coding cis-regulatory elements (CREs) that culminate in altered gene expression. Although Genome Wide Association Studies support this hypothesis, it’s still unclear how mutations on CREs contribute to disease. The translation from the “non-coding code” to phenotype is an exciting and unexplored field that we will approach in this project with the help of the zebrafish as a suitable animal model. We aim to uncover the implications of the disruption of pancreas CREs and how they contribute to diabetes in vivo. For this we will study transcriptional regulation of genes in zebrafish. The similarities between zebrafish and mammal pancreas and the evolutionary conservation of pancreas transcription factors (TF) make it an excellent model to approach and study this disease. In this project we will characterize the zebrafish insulin producing beta-cell regulome, by determining the active CREs in this cell type and their bound TFs. Then we will compare this information with a similar dataset recently available for human beta-cells, to define functional orthologs in these species. Selected CREs will be tested by in vivo gene reporter assays in zebrafish, focusing on those functionally equivalent to human CREs where risk alleles have been associated with diabetes or those regulating genes involved in diabetes. Later these CREs will be mutated in the zebrafish genome to validate their contribution to diabetes. Finally we will translate this to predict new human disease-associated CREs by focusing on the regulatory landscape of diabetes-associated genes, without the need of having countless patients to uncover them. With this project we will create a model system that will allow the identification of new diabetes-associated CREs, which might have a great impact in clinical management of this epidemic disease.